Helper-dependent adenoviral gene therapy delivery and expression system

ABSTRACT

The present invention relates to gene therapy delivery and expression systems comprising at least one helper-dependent adenoviral vector containing a nucleic acid sequence encoding for proteoglycan 4 (PRG4) or a biologically active fragment thereof. The invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one helper-dependent adenoviral vector containing said nucleic acid sequence encoding for proteoglycan 4 (PRG4), or a homolog thereof from any other species, or a biologically active fragment thereof. The invention also relates to the use of the novel gene therapy delivery and expression system according to the invention for use in the prevention and/or treatment of camptodactyly-arthropathy-coxa vara-pericarditis (CACP), or a musculoskeletal disorder such as a joint disorder or joint disease.

CROSS REFERENCE TO RELATED APPLICATIONS

In accordance with 37 C.F.R. § 176, a claim of priority is included inan Application Data Sheet filed concurrently herewith. The presentapplication is a continuation application of U.S. patent applicationSer. No. 17/358,904, filed Jun. 25, 2021, which is a continuationapplication of U.S. patent application Ser. No. 14/763,326, filed Jul.24, 2015, which is a national stage filing in accordance with 35 U.S.C.§ of International Patent Application No. PCT/IB2014/000071, filed Jan.27, 2014, which claims the benefit of U.S. Provisional PatentApplication No. 61/756,516, filed Jan. 25, 2013, the entire contents ofeach of which are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The contents of the file named “GEQB-001_03US_SeqList_ST25”, which wascreated on Jan. 13, 2023, and is 241,968 bytes, are hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to gene therapy delivery and expressionsystems comprising at least one helper-dependent adenoviral vectorcontaining a nucleic acid sequence encoding for proteoglycan 4 (PRG4) ora biologically active fragment thereof. The invention further relates toa pharmaceutical composition comprising a therapeutically effectiveamount of at least one helper-dependent adenoviral vector containingsaid nucleic acid sequence encoding for proteoglycan 4 (PRG4), or ahomolog thereof from any other species, or a biologically activefragment thereof. The invention also relates to the use of the novelgene therapy delivery and expression system according to the inventionfor use in the prevention and/or treatment ofcamptodactyly-arthropathy-coxa vara-pericarditis (CACP), or amusculoskeletal disorder such as a joint disorder or joint disease.

BACKGROUND OF THE INVENTION

Musculoskeletal conditions are the most common chronic conditions,affecting nearly one third of the human population. Musculoskeletalconditions are defined as conditions of the bones, muscles and theirattachments such as joints, tendons and ligaments. They consist of avariety of different diseases that cause pain or discomfort in thebones, joints, tendons, ligaments, muscles or surrounding structures.Musculoskeletal disorders range from back pain to rheumatoid arthritis,and gout, and include different types of arthritis, tendinitis andmusculoskeletal pain. Furthermore, musculoskeletal diseases or disordersinclude, but are not limited to arthropathies, all types of arthritis,including arthritis-related disorders, osteoarthritis, rheumatoidarthritis, gout and pseudo-gout, septic arthritis, psoriatic arthritis,ankylosing spondylitis, juvenile idiopathic arthritis. Still's disease,Reiter's syndrome, or tendinopathies including tendonitis, tendinosis,tenosynovitis; synovial disorders including synovitis; Bursa disordersincluding bursitis; equine musculoskeletal disorders including bonespavin, navicular syndrome, osselet.

In addition, there are heritable disorders such as CAPC(camptodactyly-arthropathy-coxa vara-pericarditis) syndrome that havetheir origin in a non-functional PRG4 gene. The disorder results insynoviocyte hyperplasia and early onset osteoarthritis, the principalpathological features of the CAPC syndrome.

Osteoarthritis (OA) is an age-related or post-traumatic degenerativedisease of the joint that is characterized by loss of articularcartilage, chondrocyte proliferation and hypertrophic differentiation,subchondral bone remodelling, inflammation, and finally, osteophyteformation (K. Johnson et al., A Stem Cell-Based Approach to CartilageRepair. Science (New York, N.Y.) 336, 717 (Jun. 10, 2012)). It is amongthe leading causes of chronic disability (Matthews, G. L., and Hunter,D. J. (2011), Emerging drugs for osteoarthritis. Expert Opin. EmergingDrugs 1-13). Surprisingly, given the impact of OA, relatively fewgenetic mouse models have been developed to provide insights intopotential protective mechanisms that can modify the development ofosteoarthritis. To date, most have been loss-of-function genetic modelsof cartilage degrading enzymes such as ADAMTS5 and MMP13 (F. Echtermeyeret al., Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdownin osteoarthritis. Nature Medicine, 1 (Mar. 30, 2102); T. Saito et al.,Transcriptional regulation of endochondral ossification by HIF-2α duringskeletal growth and osteoarthritis development. Nature Medicine 16, 678(Jun. 23, 2010); R. M. Borzi et al., Matrix metalloproteinase 13 lossassociated with impaired extracellular matrix remodeling disruptschondrocyte differentiation by concerted effects on multiple regulatoryfactors. Arthritis & amp; Rheumatism 62, 2370 (May 13, 2010); J. D. Kayet al., Intra-articular gene delivery and expression of interleukin-1Ramediated by self-complementary adeno-associated virus. The journal ofgene medicine 11, 605 (July, 2009)). Mice with loss of function mutationin Hif2a are also protected from osteoarthritis development,highlighting the importance of the hypoxia pathway in cartilagehomeostasis. Unfortunately, despite significant investment, thedevelopment of inhibitors of such pathways has not proven effective inthe clinical setting.

Interestingly, loss-of-function mutations in proteoglycan 4 (PRG4) inhumans cause Camptodactyly-Arthropathy-Coxa Vara-Pericarditis Syndrome(J. Marcelino et al., CACP, encoding a secreted proteoglycan, is mutatedin camptodactyly-arthropathy-coxa vara-pericarditis syndrome. Naturegenetics 23, 319 (November, 1999)), which is characterized by earlyonset osteoarthritis. In addition, genetic knockout of PRG4 in mice alsoresults in early osteoarthritis development (D. K. Rhee et al., Thesecreted glycoprotein lubricin protects cartilage surfaces and inhibitssynovial cell overgrowth, J Clin Invest 115, 622 (March, 2005); J. M.Coles et al., Loss of cartilage structure, stiffness, and frictionalproperties in mice lacking PRG4. Arthritis & amp; Rheumatism 62, 1666(Jul. 1, 2010)).

PRG4 is also known as lubricin or superficial zone protein ormegakaryocyte stimulating factor precursor. It is a component of thecartilage extracellular matrix and synovial fluid (D. K. Rhee et al.,The secreted glycoprotein lubricin protects cartilage surfaces andinhibits synovial cell overgrowth, J Clin Invest 115, 622 (March,2005)). PRG4 is present in synovial fluid and on the surface(superficial layer) of articular cartilage and therefore plays animportant role in joint lubrication and synovial homeostasis. Unlikeprevious osteoarthritis targets, it is a secreted protein produced bysuperficial zone chondrocytes of the articular cartilage and by synoviallining cells in mammals (D, K. Rhee et al., The secreted glycoproteinlubricin protects cartilage surfaces and inhibits synovial cellovergrowth. J Clin Invest 115, 622 (March, 2005)). The PRG4 gene encodesfor glycoprotein of approximately 345 kDa. PRG4 provides synovial fluidwith the ability to dissipate strain energy under load and itsrecombinant protein has been reported to exert chondroprotective effectsduring the progression of OA in rats (G. D. Jay, J. R. Torres, M. L.Warman, M. C. Lederer, K. S. Breuer, The role of lubricin in themechanical behavior of synovial fluid. Proc Natl Acad Sci USA 104, 6194(Apr. 10, 2007); C. R. Flannery et al., Prevention of cartilagedegeneration in a rat model of osteoarthritis by intraarticulartreatment with recombinant lubricin. Arthritis & amp; Rheumatism 60, 840(April, 2009)). However, the long-term biological effects of PRG4over-expression and the molecular mechanism of its potential therapeuticbenefits are still poorly understood.

U.S. Pat. No. 6,743,774 A describes a gene therapy approach byadministering to a mammal a nucleic acid encoding a therapeuticlubricating polypeptide, such as a lubricating fragment of megakaryocytestimulating factor precursor by standard vectors and/or gene deliverysystems. The gene delivery systems described include liposomes,receptor-mediated delivery systems, naked DNA, viral vectors such asherpes viruses, retro viruses, adenoviruses and adeno-associatedviruses. U.S. Pat. No. 7,393,029 A describes polynucleotides for use ingene therapy encoding for recombinant lubricin.

Although some approaches suggest gene therapy for treating or preventingjoint disorders such as osteoarthritis, no curative treatments arecurrently available. Medical treatment is mostly aimed at alleviatingthe symptoms using analgesic drugs rather than establishing worn awaycartilage. An analgesic treatment usually involves steroids andnon-steroidal anti-inflammatory drugs (NSAIDS), which have shownefficacy in the treatment of osteoarthritis for some decades. However,while these drugs can suppress joint inflammation, many of them areknown to have deteriorating effects on the cartilage, which furtherworsens the underlying process of osteoarthritis development. Hyaluronicacid, which restores viscoelasticity and lubrication of the joints, hasalso been widely used. Furthermore, polysulphated glycosaminoglycansinjected into the joint or intramuscularly as well as orallyadministered glucosamine and chondroitin sulphate have been used in thetreatment for osteoarthritis, however, the efficacy has not been provenin large randomized trials. Thus, currently used therapies have onlylimited efficacy in the treatment of joint disorders such asosteoarthritis and their success often depends on the severity of thecase. Moreover, these drugs must be administered frequently; sometimesin combination with each other. However, frequent drug injections intothe joint are laborious, bear the risk for infections, cause stress forthe patient and are costly. It follows that there is a clear and yetunmet medical need for more efficacious and sustained treatments thatare at the same time also cost effective in the long run.

The role of PRG4 in joint disorders has been discussed. In addition,during osteoarthritis, interleukin-1 (Il-1) functions as a centralmediator of inflammation (Daheshia, M., and YAO, J. Q. (2008). TheInterleukin 1β Pathway in the Pathogenesis of Osteoarthritis, JRheumatol 35, 2306). Moreover, Il-1 strongly inhibits cartilage matrixsynthesis and can trigger matrix breakdown (Evans, C. H., Gouze, J. N.Gouze, E., Robbins, P. D, and Ghivizzani, S. C. (2004)). Osteoarthritisgene therapy, Gene Ther 11, 379-389). To neutralize the effect of Il-1on synovial inflammation, treatment with interleukin-1 receptorantagonist (Il-1Ra) constitutes a promising concept in the therapy ofosteoarthritis (Evans, C. H., Gauze, J. N., Gauze, E., Robbins, P. D.,and Ghivizzani. S. C. (2004). Osteoarthritis gene therapy. Gene Ther 11,379-389; Caron J P et al. Chondroprotective effect of intraarticularinjections of interleukin-1 receptor antagonist in experimentalosteoarthritis. Suppression of collagenase-1 expression. Arthritis Rheum1996; 39: 1535-1544)). On nucleic acid level, Il-1Ra is considerablyconserved among mammalian species. For example, the cDNA sequences ofhuman Il-1Ra (Accession no: NM_173842) shares 82% homology with themurine variant (Accession no: NM_031167), 84% with the equine variant(Accession no: NM_001082525), 84% with the canine variant (Accession no:NM_001003096), 84% with the lapine variant (Accession no: NM_001082770)and 82% with the bovine variant (Accession no: NM_174357).

Although gene therapy approaches using various gene therapy vectors areknown, there is a need for a gene therapy delivery and expressionsystem, which allows for the specific delivery of a therapeutic amountof an active agent to its target. In addition, the active agent shallexhibit its therapeutic effects for a prolonged amount of time.Adeno-associated viruses (AAV) are among the most widely used genetherapy vectors and have shown efficient transduction and long-termtransgene expression in many tissues. AAVs have also been used in genetherapy approaches for joints. However, AAV transduction efficiency injoints has never been directly compared to transduction efficiency ofother viral gene therapy vectors such as adenoviruses includinghelper-dependent adenoviral vectors.

Helper-dependent adenoviruses (HDAd), also known as gutless orhigh-capacity adenoviruses, are the latest generation of adenoviralvectors (Mitani, K., Graham, F. L. Caskey, C. T. & Kochanek, S. Rescue,propagation, and partial purification of a helper virus-dependentadenovirus vector. Proc Natl Acad Sci USA 92, 3854-3858 (1995); Parks,R. J. et al. A Helper-dependent adenovirus vector system: removal ofhelper virus by Cre-mediated excision of the viral packaging signal.Proc Natl Acad Sci USA 93, 13565-13570 (1996); Parks, R. J. Improvementsin adenoviral vector technology: overcoming barriers for gene therapy.Clin. Genet. 58, 1-11 (2000)). These vectors are devoid of all viralsequences and are able to mediate long-term gene expression in varioustissues (e.g. 7 years in the liver) in contrast to the more immunogenicfirst generation adenoviruses (Brunetti-Pierri, N., Ng, T., Iannitti,D., Cioffi, W., Stapleton, G., Law, M., Breinholt, J., Palmer; D.,Grove, N., Rice, K., et al. (2013). Transgene Expression up to 7 Yearsin Nonhuman Primates Following Hepatic Transduction withHelper-Dependent Adenoviral Vectors. Hum Gene Ther 24, 761-765).

BRIEF SUMMARY OF THE INVENTION

In one aspect, provided herein is a gene therapy delivery and expressionsystem, comprising at least one helper-dependent adenoviral vectorcontaining a nucleic acid sequence encoding proteoglycan 4 (PRG4), or abiologically active fragment thereof, which has chondoprotectiveactivity, left and right adenoviral inverted terminal repeats (L ITR andR ITR), adenoviral packaging signal sequences and non-viral, non-codingstuffer nucleic acid sequences, wherein PRG4 expression in the at leastone helper-dependent adenoviral vector is controlled by a ubiquitous,constitutive promoter, wherein (i) the helper-dependent adenoviralvector additionally comprises a nucleic acid sequence encodinginterleukin-1 receptor antagonist (Il-1Ra), or (ii) the delivery andexpression system comprises a second helper-dependent adenoviral vectorcomprising a nucleic acid sequence encoding interleukin-1 receptorantagonist (Il-1Ra).

In some embodiments, the ubiquitous constitutive promoter is selectedfrom the group consisting of elongation factor 1 alpha (EF1 alpha)promoter, cytomegalovirus (CMV) promoter, beta-actin promoter, simianvirus 40 (SV4O) early promoter, ubiquitin c promoter, glyceraldehyde3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase(PGK) promoter.

In some embodiments, the helper-dependent adenoviral vector containing anucleic acid sequence encoding proteoglycan 4 (PRG4) comprises a nucleicacid sequence set forth in SEQ ID NO 1, or SEQ ID NO 2, or abiologically active fragment thereof, or a nucleic acid sequence whichhas at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleicacid sequence set forth in SEQ ID NO 1, or SEQ ID NO 2, or abiologically active fragment thereof, wherein the helper-dependentadenoviral vector containing a nucleic acid sequence encoding PRG4,which comprises a biologically active fragment of SEQ ID NO 1, or SEQ IDNO 2, or a nucleic acid sequence which has at least 50%, 60%, 70%, 80%or 90% sequence homology with a nucleic acid sequence set forth in SEQID NO 1. or SEQ ID NO 2, or a biologically active fragment thereof,mediates chondoprotective activity.

In some embodiments, the nucleic acid sequence encoding proteoglycan 4(PRG4) comprises a nucleic acid sequence set forth in SEQ ID NO 3, orSEQ ID NO 4, or a biologically active fragment thereof, or a homologthereof from any other species, or a nucleic acid sequence which has atleast 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acidsequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or a biologicallyactive fragment thereof, wherein the PRG4 encoded by the biologicallyactive fragment of SEQ ID NO 3, or SEQ ID NO 4, or a nucleic acidsequence which has at least 50%, 60%, 70%, 80% or 90% sequence homologywith a nucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4,or a biologically active fragment thereof, has chondoprotectiveactivity.

In some embodiments, the amino acid sequence of proteoglycan 4 (PRG4)comprises an amino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO6, or a biologically active fragment thereof, or a homolog thereof fromany other species, or an amino acid sequence which has at least 50%,60%, 70%, 80% or 90% sequence homology with an amino acid sequence setforth in SEQ ID NO 5, or SEQ ID NO 6, or a biologically active fragmentthereof, wherein the amino acid sequence of PRG4, which comprises abiologically active fragment of the amino acid SEQ ID NO 5, or SEQ ID NO6, or has at least 50%, 60%, 70%, 80% or 90% sequence homology with anamino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or abiologically active fragment thereof, has chondoprotective activity.

In some embodiments, expression of interleukin-1 receptor antagonist(Il-1Ra) is controlled by an inflammation-inducible promoter selectedfrom the group consisting of NF-κB promoter, interleukin 6 (Il-6)promoter, interleukin-1 (Il-1) promoter, tumor necrosis factor (TNF)promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3)promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatoryprotein-1α (MIP-1α) promoter, or hybrid constructs of the above.

In some embodiments, the helper-dependent adenoviral vector comprisingthe nucleic acid sequence encoding interleukin-1 receptor antagonist(Il-1Ra), comprises a nucleic acid sequence which has at least 50%, 60%,70%, 80% or 90% sequence homology with a nucleic acid sequence set forthin SEQ ID NO 7, or SEQ ID NO 8, or SEQ ID NO 9, or a biologically activefragment thereof, wherein the helper-dependent adenoviral vectorcomprising the nucleic acid sequence encoding Il-1Ra, which comprises anucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90%sequence homology with a nucleic acid sequence set forth in SEQ ID NO 7,or SEQ ID NO 8, or SEQ ID NO 9, or a biologically active fragmentthereof, has Il1-Ra activity of inhibiting inflammatory and cartilagedestructive mediators.

In some embodiments, the nucleic acid sequence encoding interleukin-1receptor antagonist (Il-1Ra) comprises a nucleic acid sequence set forthin SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, or a biologicallyactive fragment thereof, or wherein the nucleic acid sequence encodingfor interleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acidsequence which has at least 50%, 60%, 70%, 80% or 90% sequence homologywith a nucleic acid sequence set forth in SEQ ID NO 10, or SEQ ID NO 11,or SEQ ID NO 12, or a biologically active fragment thereof, wherein theIl-1Ra encoded by the biologically active fragment of SEQ ID NO 10, orSEQ ID NO 11, or SEQ ID NO 12, or the nucleic acid sequence which has atleast 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acidsequence set forth in SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, orthe biologically active fragment thereof, mediates Il1-Ra activity ofinhibiting inflammatory and cartilage destructive mediators.

In some embodiments, the amino acid sequence of interleukin-1 receptorantagonist (Il-1Ra) comprises an amino acid sequence set forth in SEQ IDNO 13, SEQ ID NO 14, SEQ ID NO 15, or a biologically active fragmentthereof, which has Il1-Ra activity of inhibiting inflammatory andcartilage destructive mediators.

In another aspect, provided herein is a pharmaceutical composition,comprising a therapeutically effective amount of at least onehelper-dependent adenoviral vector containing a nucleic acid sequenceencoding proteoglycan 4 (PRG4), or a biologically active fragmentthereof, which has chondoprotective activity, left and right adenoviralinverted terminal repeats (L ITR and R ITR), adenoviral packaging signalsequences and non-viral, non-coding stuffer nucleic acid sequences,wherein PRG4 expression in the at least one helper-dependent adenoviralvector is controlled by a ubiquitous, constitutive promoter, wherein (i)the helper-dependent adenoviral vector additionally comprises a nucleicacid sequence encoding interleukin-1 receptor antagonist (Il-1Ra), or(ii) the delivery and expression system comprises a secondhelper-dependent adenoviral vector comprising a nucleic acid sequenceencoding interleukin-1 receptor antagonist (Il-1Ra).

In some embodiments, the ubiquitous constitutive promoter is selectedfrom the group consisting of elongation factor 1 alpha (EF 1 alpha)promoter, cytomegalovirus (CMV) promoter, beta-actin promoter, simianvirus 40 (SV40) early promoter, ubiquitin c promoter, glyceraldehyde3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase(PGK) promoter.

In some embodiments, wherein the helper-dependent adenoviral vectorcontaining a nucleic acid sequence encoding proteoglycan 4 (PRG4)comprises a nucleic acid sequence set forth in SEQ ID NO 1, or SEQ ID NO2, or a biologically active fragment thereof, or a nucleic acid sequencewhich has at least 50%, 60%, 70%, 80% or 90% sequence homology with anucleic acid sequence set forth in SEQ ID NO 1, or SEQ ID NO 2, or abiologically active fragment thereof, wherein the helper-dependentadenoviral vector containing a nucleic acid sequence encoding PRG4,which comprises a biologically active fragment of SEQ ID NO 1, or SEQ IDNO 2, or a nucleic acid sequence which has at least 50%, 60%, 70%, 80%or 90% sequence homology with a nucleic acid sequence set forth in SEQID NO 1. or SEQ ID NO 2, or a biologically active fragment thereof,mediates chondoprotective activity.

In some embodiments, the nucleic acid sequence encoding proteoglycan 4(PRG4) comprises a nucleic acid sequence set forth in SEQ ID NO 3, orSEQ ID NO 4, or a biologically active fragment thereof, or a homologthereof from any other species, or a nucleic acid sequence which has atleast 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acidsequence set forth in SEQ ID NO 3, SEQ ID NO 4, or a biologically activefragment thereof, wherein the PRG4 encoded by the biologically activefragment of SEQ ID NO 3, or SEQ ID NO 4, or a nucleic acid sequencewhich has at least 50%, 60%, 70%, 80% or 90% sequence homology with anucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or abiologically active fragment thereof, has chondoprotective activity.

In some embodiments, the amino acid sequence of proteoglycan 4 (PRG4)comprises an amino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO6, or a biologically active fragment thereof, or a homolog thereof fromany other species, or an amino acid sequence which has at least 50%,60%, 70%, 80% or 90% sequence homology with an amino acid sequence setforth in SEQ ID NO 5, or SEQ ID NO 6, or a biologically active fragmentthereof, wherein the amino acid sequence of PRG4, which comprises abiologically active fragment of the amino acid SEQ ID NO 5, or SEQ ID NO6, or has at least 50%, 60%, 70%, 80% or 90% sequence homology with anamino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or abiologically active fragment thereof, has chondoprotective activity.

In some embodiments, expression of interleukin-1 receptor antagonist(Il-1Ra) is controlled by an inflammation-inducible promoter selectedfrom the group consisting of NF-κB promoter, interleukin 6 (Il-6)promoter, interleukin-1 (Il-1) promoter, tumor necrosis factor (INF)promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3)promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatoryprotein-1α (MIP-1α) promoter, or hybrid constructs of the above.

In some embodiments, the helper-dependent adenoviral vector comprisingthe nucleic acid sequence encoding interleukin-1 receptor antagonist(Il-1Ra), comprises a nucleic acid sequence which has at least 50%, 60%,70%, 80 or 90% sequence homology with a nucleic acid sequence set forthin SEQ ID NO 7, or SEQ ID NO 8, or SEQ ID NO 9, or a biologically activefragment thereof, wherein the helper-dependent adenoviral vectorcomprising the nucleic acid sequence encoding Il-1Ra, which comprises anucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90%sequence homology with a nucleic acid sequence set forth in SEQ ID NO 7,or SEQ ID NO 8, or SEQ ID NO 9, or a biologically active fragmentthereof, has Il1-Ra activity of inhibiting inflammatory and cartilagedestructive mediators.

In some embodiments, the nucleic acid sequence encoding interleukin-1receptor antagonist (Il-1Ra) comprises a nucleic acid sequence set forthin SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, or a biologicallyactive fragment thereof, or wherein the nucleic acid sequence encodingfor interleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acidsequence which has at least 50%, 60%, 70%, 80% or 90% sequence homologywith a nucleic acid sequence set forth in SEQ ID NO 10, or SEQ ID NO 11,or SEQ ID NO 12, or a biologically active fragment thereof, wherein theIl-1Ra encoded by the biologically active fragment of SEQ ID NO 10, orSEQ ID NO 11, or SEQ ID NO 12, or the nucleic acid sequence which has atleast 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acidsequence set forth in SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, orthe biologically active fragment thereof, mediates Il1-Ra activity ofinhibiting inflammatory and cartilage destructive mediators.

In some embodiments, the amino acid sequence of interleukin-1 receptorantagonist (Il-1Ra) comprises an amino acid sequence set forth in SEQ IDNO 13, SEQ ID NO 14, SEQ ID NO 15, or a biologically active fragmentthereof, which has Il1-Ra activity of inhibiting inflammatory andcartilage destructive mediators.

In another aspect, provided herein is the gene therapy delivery andexpression system according to any of the preceding embodiments for usein the prevention and/or treatment of camptodactyly-arthropathy-coxavara-pericarditis (CACP) syndrome, or a musculoskeletal disorder, or ajoint disorder or disease. In some embodiments, the disease or disorderis selected from the group consisting of arthropathies, all types ofarthritis, including arthritis-related disorders, osteoarthritis,rheumatoid arthritis, gout and pseudo-gout, septic arthritis, psoriaticarthritis, ankylosing spondylitis, juvenile idiopathic arthritis,Still's disease, Reiter's syndrome, or tendinopathies includingtendonitis, tendinosis, tenosynovitis; synovial disorders includingsynovitis; Bursa disorders including bursitis; equine musculoskeletaldisorders including bone spavin, navicular syndrome, osselet.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Prg4 transgenic mice are protected from development ofage-related osteoarthritis. A, Comparison of 10 month-old wild type miceand Prg4 transgenic mice knee joints by OARSI grade (*P<0.05, n=7.Wilcox rank test). B, Safranin O staining and immunohistochemistry(antibody used to the left) of 10 month-old wild type (Wt) and Prg4transgenic mice. Black arrows indicate osteoarthritis changes inproximal tibial articular cartilage in sagittal section, Scale bar, 100pm μm. C, Representative image of the reconstruction of articularcartilage in 10-month-old wild type and Prg4 transgenic mouse withfemoral cartilage shown in blue and tibial cartilage shown in yellow.Red arrow indicates loss of cartilage. Scale bar, 500 μm. D,Quantification of articular cartilage volume and surface area of bonecovered by cartilage in mouse knee joints by phase contrast μCT.(*P<0.01, n=5, t-test). Error bars indicate s.e.m.

FIG. 2 . Prg4 transgenic mice are protected from the development ofpost-traumatic osteoarthritis, A, Comparison of Prg4 transgenic mice andwild type mice knee joints by OARSI grade (*P<0.05, n=8, ANOVA). B,Safranin O staining and immunohistochemistry (antibody used listed aboveeach column) of wild type sham (Wt Sham), wild type with transection (WtSx) and Prg4 transgenic mice with transection (Prg4 Sx). Black arrowsindicate areas with osteoarthritis changes in saggital sections throughthe knee. Scale bar, 100 μm. C, Representative images of reconstructionof knee joints with phase contrast μCT with femoral cartilage shown inblue and tibial cartilage shown in yellow. Red arrow indicates loss ofcartilage, Scale bar, 500 μm. D, Quantification of articular cartilagevolume and surface area of bone covered by cartilage in mouse joints byphase contrast μCT (*P<0.01, **P<0.05, N.S.=not significant, n=5-6,ANOVA). E, Average time that mice stayed on rotating rod 2 months aftercruciate ligament transection in rotarod analysis (*P<0.05, n=15,ANOVA). F, Response time of mice after placement onto a 55° C. platformin hotplate analysis (*P<0.05, n=10, ANOVA), Error bars indicate s.e.m.

FIG. 3 : PRG4 delivered by helper-dependent adenoviral vectors (HDAd)protects mice from development of osteoarthritis. A, Representativeimage comparing intra-articular injection of HDAd and differentserotypes of AAV. Vectors (10⁹ viral particles) each expressing GFP wereinjected intraarticularly into the knee joint. The lower panels areenlarged images of the boxed areas in the upper panels. Green: GFP,blue: DAPI. C: cartilage, S: synovium. Scale bar, 100 μm. B, Comparisonof luciferase expression after intra-articular injection offirst-generation adenoviral vector (FGV) and helper-dependent adenoviralvector (HDAd) (n=6) into the knee joint. C, Representative image ofexpression patterns of intra-articular injections of different doses ofHDAD encoding □-galactosidase. The bottom images are enlarged areas ofthe black box in the upper images of saggital sections through the kneejoint. C: Cartilage; S: Synovium. Scale bar, 100 μm. D-G, Scheme ofexperiment comparing the preventive effect HDAd-PRG4 injection andevaluation by OARSI grade and cartilage volume (D). Before transection,wild type mice were injected with HDAd-PRG4 intra-articularly at 10⁹vp/joint (Sx+PRG4 H) or 10⁸ vp/joint (Sx+PRG4 L). Sham, transectionwithout treatment (Sx) and injection of virus without transgene beforetransection (Sx+Vector) served as controls. Degree of osteoarthritis ispresented by OARSI grade (E), cartilage volume (F) and cartilage surfacearea (G) (*P<0.05, n=8-10 in OARSI grading, n=5-6 in cartilage volumeanalysis, ANOVA). H-K, Scheme of experiment comparing the protectiveeffect HDAd-PRG4 injection and evaluation by OARSI grade and cartilagevolume (H). Two weeks after transection, wild type mice were injectedwith HDAd-PRG4 intra-articularly at 10⁹ vp/joint (Sx+PRG4 L) or 10⁸vp/joint (Sx+PRG4 L), Sham, no treatment (Sx) and HDAd-GFP injection(Sx+GFP) served as controls. Degree of OA is presented by OARSI grade(I), cartilage volume (J) and cartilage surface area (K) (*P<0.05,n=8-10 in OARSI grading, n=5-6 in cartilage volume analysis, ANOVA).Error bars indicate s.e.m.

FIG. 4 : PRG4 delays osteoarthritis by inhibiting cartilage catabolismand terminal hypertrophy. A, Microarray heat map analysis comparingsuperficial zone cartilage of wild type and Prg4 transgenic mice, Geneswith expression changes larger than 1.5 fold and p-value less than 0.05are plotted. B, Transcription factor activity changes predicted byIngenuity Pathway Analysis. All transcription factors shown here werepredicted to be suppressed by Ingenuity Pathway Analysis. X-axisindicates the number of genes/gene groups controlled by eachtranscription pathway in the gene list submitted. C, Changes in geneexpression (Prg4, Hif3a, Vegf, Col10a1 and Mmp13) in C3H10T1/2 cellsunder hypoxia (1% oxygen for 8 hours). Cells are sham treated, infectedwith empty HDAd (vector) or with HDAd-PRG4 (PRG4) (*P<0.05, n=3, ANOVA).D, Changes of PRG4 and Hif3alpha expression under normoxia and hypoxiain TC71 Ewing sarcoma cells (*P<0.05, n=3, t-test). E, Changes of geneexpression after Hif3alpha knockdown by siRNA (*P<0.05, n=3, ANOVA). F,Proposed model of PRG4 function in prevention of osteoarthritisdevelopment. Error bars indicate s.e.m.

FIG. 5 : PRG4 over-expression under the Col2al promoter does notadversely affect the development of mice. A, Schematic figure of thePrg4 transgenic mice construct. B, Rib cartilage expression levels ofPRG4, Sox9 and Col2al in newborns (P1) PRG4 transgenic and wild typemice (*P<0.05, n=7-8, student t-test). C, Comparison of anti-PRG4antibody stained femoral head section in wild type and Prg4 transgenicnewborn mice. Scale bars, 100 μm. D, Comparison of anti-PRG4 antibodystained knee joints in Prg4 transgenic and wild type 3 month-old mice.The boxed areas to the left (articular area, distal femoral growth plateand proximal tibial growth plate) are enlarged at the right. Scale bars,100 μm. E, Weight of Prg4 high expresser, low expresser and wild typemice (P<0.05, n=10-11). F, Representative image of BrdU (red) and DAPI(blue) staining in wild type and Prg4 transgenic P3 hind limbs. Stainingsignals were quantified by image J (P<0.05, n=7). Scale bars, 100 μm. G,Representative image of TUNEL (brown) and methyl green staining in wildtype and Prg4 transgenic P3 hind limbs. No positive signals were visiblein cartilage but they were present in overlying dermis and skin. Scalebar, 100 μm. Error bars indicate s.e.m.

FIG. 6 : Generation and characterization of HDAd-PRG4. A, HDAd-PRG4 bandis visible in the last step of cesium chloride ultracentrifugation. Thepurified virus is the white band in the tube. B, PCR determination ofPRG4 expression of HEK293 cells infected with HDAd-PRG4 (PRG4), HDAd-GFP(GFP), and sham treatment (cells). Gapdh is used as loading control. C,Immunofluorescence of HEK293 cells infected with HDAd-PRG4 (PRG4, red)and HDAd-GFP (GFP, green). DAPI (blue) was used as counter-stain.

FIG. 7 : Gene expression profiling of wild type vs. Prg4 transgenicsuperficial layer chondrocytes. A, Laser capture micro-dissection ofsuperficial layer chondrocytes. Left to right: tissue before capturing,captured tissue on HS cap, and remaining tissue on the slide. Scale bar,100 μm. B, Evaluation of Hif3a expression in superficial layerchondrocytes of wild type vs. Prg4 mice (*Py0.05, n=3, t-test). C,Evaluation of PRG4 expression in ATDC5 and C3H10T1/2 cells undernormoxic and hypoxic conditions (*P<0.05, n=3, t-test), Error barsindicate s.e.m.

FIG. 8 : A, Mice were treated with HDAd-PRG4 (PRG4), HDAd-Il-1Ra (Il1Ra)and combination therapy (PRG4+Il1Ra) according to the scheme shown inFIG. 3 H. All three groups showed significantly lower histological scorecompared to no treatment and placebo treatment, suggesting therapeuticeffects to HDAd-PRG4 and HDM-Il-1Ra. The combination therapy showed atrend of being more effective, (ANOVA, N=8-10, *p<0.05); B, C, Mice weretreated with HDAd-PRG4 (PRG4), HDAd-Il1-Ra (Il1Ra) and combinationtherapy according to the scheme shown in FIG. 3 H. Cartilage surfacearea (B) and cartilage volume (C) were quantified. All three groupsshowed significantly higher cartilage volume and surface area comparedto no treatment and placebo treatment, suggesting therapeutic effects ofHDAd-PRG4 and HDAd-Il1Ra. The knees under combination therapy had morepreserved cartilage volume and surface area (ANOVA, N=5, *p<0.05).

DETAILED DESCRIPTION

It is therefore an object of the present invention to provide animproved delivery and expression system that allows for long-termexpression of biologically active proteoglycan 4 (PRG4) for use in theprevention and/or treatment of PRG4-dependent disorders such ascamptodactyly-arthropathy-coxa vara-pericarditis (CACP) and disorders inwhich PRG4 overexpression is beneficial such as musculoskeletaldisorders in particular joint disorders.

The solution for the problem is provided by a gene therapy delivery andexpression system, comprising the technical features as claimed in claim1. Preferred embodiments of the invention are subject-matter of thedependent claims.

The gene therapy delivery and expression system according to the presentinvention comprises at least one helper-dependent adenoviral vectorcontaining a nucleic acid sequence encoding for proteoglycan 4 (PRG4),or a biologically active fragment thereof, left and right adenoviralinverted terminal repeats (L ITR and R ITR), adenoviral packaging signalsequences and non-viral, non-coding stuffer nucleic acid sequences.

Any known left or right adenoviral inverted terminal repeats (L ITR andR ITR), adenoviral packaging signal sequences and non-viral, non-codingstuffer nucleic acid sequences can be used for the production of thehelper-dependent adenoviral vector (Parks, R. J., Chen, L., Anton, M.,Sankar, U., Rudnicki, M. A., and Graham, F. L. (1996). Ahelper-dependent adenovirus vector system: removal of helper virus byCre-mediated excision of the viral packaging signal. Proc Natl Acad SciUSA 93, 13565-13570; Palmer, D. and Ng, P. (2003). Improved system forhelper-dependent adenoviral vector production. Mol Ther 8, 846-852).

The results and data shown herein are based on helper-dependentadenoviral constructs (HDAd) using in a first embodiment nucleic acidsequences or amino-acid sequences encoding for proteoglycan 4 (PRG4).Any homolog or variant showing a certain degree of sequence homologywith either a nucleic acid sequence or an amino-acid sequence ofproteoglycan 4 (PRG4) (or their variant such as lubricin, superficialzone protein or megakaryocyte stimulating factor precursor) is furthercomprised by the present invention. A homolog includes but is notlimited to peptides, polypeptides, proteins or nucleic acid sequencesfrom any species that shows homology with any proteoglycan (PRG4)described herein,

For long-term expression of PRG4 in the affected tissue, for example injoints or osteoarthritic tissues, the at least one helper-dependentadenoviral vector of the invention is preferably controlled by aubiquitous, constitutive promoter. Suitable promoters include, but arenot limited to elongation factor 1 alpha (EF1 alpha) promoter,cytomegalovirus (CMV) promoter, beta-actin promoter, simian virus 40(SV40) early promoter, ubiquitin c promoter, glyceraldehyde 3-phosphatedehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoterand other HDAd-suitable ubiquitous, constitutive promoters.

In a preferred embodiment, the helper-dependent adenoviral vectorcomprising proteoglycan 4 (PRG4) comprises a nucleic acid sequence setforth in SEQ ID NO 1 (human HDAd) or SEQ ID NO 2 (murine HDAd).Preferably, the nucleic acid sequence comprises a cDNA sequence of thePRG4 gene or a fragment thereof. Furthermore, any biologically activefragment such as nucleic acid sequences having sequence identity or acertain degree of homology with the human or murine PRG4 sequencedisclosed herein is comprised by the present invention. The HDAd of theinvention may vary in its non-coding elements as well as in the lengthof its coding insert. Therefore, also smaller or greater vector sizes ofthe helper-dependent adenoviral vector of the invention can be utilizedfor the purpose of the present invention in order to achieve the desiredbiological effects.

In a preferred embodiment, the helper-dependent adenoviral vectorcomprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80%or 90% sequence homology with a vector sequence comprising a nucleicacid sequence set forth in SEQ ID NO 1 or SEQ ID NO 2, or a biologicallyeffective fragment thereof.

A sequence homology of at least 50% with a nucleic acid sequence setforth in SEQ ID NO 1 or SEQ ID NO 2 can be sufficient in order togenerate long-term expression of PRG4 at the target sites as along asthe expressed PRG4 protein is biologically active.

In one embodiment the helper-dependent adenoviral vector comprises anucleic sequence encoding for proteoglycan 4 (PRG4). The expression ofhuman or mammalian PRG4 is preferred. The inserted nucleic acid sequenceinto the HDAd can be anyone, which shows sequence identity or sequencehomology with a nucleic acid sequence set forth in SEQ ID NO 3 or SEQ IDNO 4. For the purpose of expression, it can be sufficient that only apart of the nucleic acid sequence set forth in SEC ID NO 3 or SEQ ID NO4, or an extended version is sufficient for the generation of the vectorand its biological activity. The biological activity can be measured byinvestigating chondoprotection.

Furthermore, also mutants, variants and homologs containing nucleic acidreplacements within the amino acid or nucleic acid sequence ofproteoglycan 4 (PRG4) are comprised by the present invention. Inparticular, the invention comprises a homolog of PRG4 from any otheranimal species having sequence homology with a sequence set forth in SEQID NO 3 or SEQ ID NO 4. For example, a homolog containing a nucleic acidsequence encoding for proteoglycan 4 (PRG4) preferably comprises anucleic acid sequence, which has at least 50%, 60%, 70%, 80% or 90%sequence homology with a nucleic acid sequence set forth in SEQ ID NO 3,or SEQ ID NO 4, or a biologically effective fragment thereof.

The proteoglycan 4 (PRG4) used in the construction of the vector of thepresent invention can be also described by its amino acid sequence.Preferably, the proteoglycan 4 (PRG4) comprises an amino acid sequenceset forth in SEQ ID NO 5 or SEQ ID NO 6, or a biologically activefragment thereof, or a homolog thereof from any other species. In apreferred embodiment, the amino acid sequence encoding for proteoglycan4 (PRG4) comprises an amino acid sequence which has at least 50%, 60%,70%, 80% or 90% sequence homology with an amino acid sequence set forthin SEQ ID NO 5, or SEQ ID NO 6, or a biologically active fragmentthereof, or a homolog thereof from any other species.

The inventors show herein that a helper-dependent adenoviral vectorcontaining the cDNA sequence of murine or human proteoglycan 4 (PRG4)under the control of a ubiquitous, constitutive promoter results in anoverexpression of PRG4 in joints, which results in protection of micefrom osteoarthritis. Since overexpression of PRG4 can be beneficial inmany other diseases, the helper-dependent vector of the invention can beused for the manufacture of a medicament for the treatment and/orprevention of a variety musculoskeletal diseases and PRG4-dependentdiseases. In particular, musculoskeletal disorders, which benefit fromPRG4 over-expression would be characterised as those disorders wherehigh concentrations of PRG4 confer a therapeutic or preventive effect.PRG4-dependent diseases or disorders would be characterised in thatnatural PRG4 expression is limited or inhibited, or in thatintracellular or extracellular PRG4 RNA or protein levels aresignificantly reduced.

The inventors compared joint transduction efficiency of helper-dependentadenoviral vectors with different AAV serotype that had been reported byothers to be useful in joint gene therapy approaches. Surprisingly, theinventors found that helper-dependent adenoviral vectors showed superiortransduction compared with all tested AAV serotypes resulting intransduction of synoviocytes and chondrocytes. The inventors furthershowed that helper-dependent adenoviral vectors allow for transgeneexpression that persists for an extended period, whereby the need forrepeated administrations of the medicament to an object suffering from adisease in which overexpression of PRG4 may be beneficial will begreatly reduced. In a model of osteoarthritis in mice, the inventorsdemonstrated that treatment of osteoarthritic joints with the HDAd-PRG4results in lower osteoarthritis histology scores, indicating that PRG4exhibits its biological effect by protecting the subject frompost-traumatic osteoarthritis. Protection against osteoarthritis withHDAd-PRG4 was demonstrated using two different schemes. In the firstscheme mice were injected with HDAd-PRG4 before osteoarthritis wasinduced. The results of this experiment show that HDAd-PRG4 can be usedin the prevention of osteoarthritis. In the second scheme,osteoarthritis was induced before HDAd-PRG4 was injected. The results ofthis experiment demonstrate that HDAd-PRG4 can be used in the treatmentof (pre-existing) osteoarthritis. In support of the biological effectsof PRG4 in joints, experiments using Prg4-transgenic mice revealed aprotection of the subjects against the development of osteoarthritiswithout other bone phenotypes.

Thus, overexpression of PRG4 under the control of a suitable ubiquitous,constitutive promoter in a HDAd allows for prevention and/or treatmentof a variety of musculoskeletal diseases such as arthropathies, alltypes of arthritis, including arthritis-related disorders,osteoarthritis, rheumatoid arthritis, gout and pseudo-gout, septicarthritis, psoriatic arthritis, ankylosing spondylitis, juvenileidiopathic arthritis. Still's disease, Reiter's syndrome, ortendinopathies including tendonitis, tendinosis, tenosynovitis; synovialdisorders including synovitis; Bursa disorders including bursitis;equine musculoskeletal disorders including bone spavin, navicularsyndrome, osselet.

Surprisingly, the inventors further found that a combined expression ofPRG4 as induced by HDAd-PRG4 and inhibitors of inflammatory andcartilage destructive mediators results in beneficial therapeutic andprotective effects. In particular, a combination of expressing of PRG4and expressing interleukin-1 receptor antagonist (Il-1Ra) resulted in anincreased efficiency in the treatment and/or prevention ofmusculoskeletal disorders.

In a preferred embodiment, the helper-dependent adenoviral vectortherefore comprises a nucleic acid sequence encoding for inhibitors ofinflammatory and cartilage destructive mediators such as cytokinesincluding Il-1, TNFa, Il-6, Il-7 Il-8, Il-11, Il-15, Il-17, Il-18,Il-21, leukemia inhibitory factor (LIF), oncostatin M; matrixmetalloproteases including MMP-1,3,9,13; aggrecanases includingADAMTS-1,4,5; toll-like receptors (TLR) such as TLR2, TLR4; and nuclearfactor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB).

In a preferred embodiment, the inhibitor of inflammatory and cartilagedestructive mediator is interleukin-1 receptor antagonist (Il-1Ra). Acombination of overexpression of PRG4 and Il-1Ra is beneficial for thetreatment and/or prevention of the diseases mentioned therein. In afirst embodiment, the cDNA sequence of interleukin-1 receptor antagonistcan be contained in the same helper-dependent adenoviral vector, whichcontains the cDNA sequence encoding for proteoglycan 4 (PRG4). In afurther embodiment, the cDNA sequence of interleukin-1 receptorantagonist can be contained in a second helper-dependent adenoviralvector, which only contains the cDNA sequence encoding for Il-1Ra.

The delivery and expression system of the invention can thereforecomprise a second or further helper-dependent adenoviral vectorcomprising a nucleic acid sequence encoding for inhibitors ofinflammatory and cartilage destructive mediators such as interleukin-1receptor antagonist (Il-1Ra).

In a preferred embodiment, the cDNA of the inhibitor of inflammatory andcartilage destructive mediator (e.g. Il1-Ra cDNA) inserted into HDAd iscontrolled by an inflammation-inducible promoter. Preferred promotersinclude, but are not limited to promoters selected from the groupconsisting of NF-κB promoter, interleukin 6 (Il-6) promoter,interleukin-1 (Il-1) promoter, tumor necrosis factor (TNF) promoter,cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter,serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1α(MIP-1α) promoter, or hybrid constructs of the above. The use of NF-κBpromoter in HDAd for the purpose of an inflammation-dependent expressionof Il-1Ra at the target sites is preferred.

In a preferred embodiment, the helper-dependent adenoviral vectorcontaining the interleukin-1 receptor antagonist (Il-1Ra) comprises anucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90%sequence homology with a nucleic acid sequence set forth in SEQ ID NO 7(human Il-1Ra), or SEQ ID NO 8 (murine Il-1Ra), SEQ ID NO 9 (equineIl-1Ra), or a biologically effective fragment thereof.

In a further embodiment, the nucleic acid sequence encoding forinterleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acidsequence set forth in SEQ ID NO 10 (human Il-1Ra), or SEQ ID NO 11(murine Il-1Ra), or SEQ ID NO 12 (equine Il-1Ra), or a biologicallyactive fragment thereof, or a homolog thereof from any other species.

In a preferred embodiment, the amino acid sequence encoding forinterleukin-1 receptor antagonist (Il-1Ra) comprises an amino acidsequence set forth in SEQ ID NO 13 (human Il-1Ra), or SEQ ID NO 14(murine Il-1Ra), or SEQ ID NO 15 (equine Il-1Ra), or a biologicallyactive fragment thereof, or a homolog thereof from any other species.

The present invention also relates to a pharmaceutical composition,comprising a therapeutically effective amount of at least onehelper-dependent adenoviral vector containing a nucleic acid sequenceencoding for proteoglycan 4 (PRG4), or a biologically active fragmentthereof. Preferred embodiments of the pharmaceutical compositioncomprise helper-dependent adenoviral vectors or a combination ofdifferent helper-dependent adenoviral vectors comprising features asdescribed above in more detail.

The gene therapy delivery and expression system according to the presentinvention is suitable for the preparation of a medicament for the use inthe prevention and/or treatment of a variety of PRG4-dependent diseases.In a first embodiment the gene therapy delivery and expression system isused in the treatment and/or prevention ofcamptodactyly-arthropathy-coxa vara-pericarditis (CACP). Thehelper-dependent adenoviral vectors of the invention can further be usedin the prevention and/or treatment of a musculoskeletal disorder, inparticular the prevention and/or treatment of a joint disorder ordisease. Examples of such diseases are arthropathies, all types ofarthritis, including arthritis-related disorders, osteoarthritis,rheumatoid arthritis, gout and pseudo-gout, septic arthritis, psoriaticarthritis, ankylosing spondylitis, juvenile idiopathic arthritis,Still's disease, Reiter's syndrome, or tendinopathies includingtendonitis, tendinosis, tenosynovitis; synovial disorders includingsynovitis; Bursa disorders including bursitis; equine musculoskeletaldisorders including bone spavin, navicular syndrome, osselet.

The following examples show the beneficial therapeutic uses of the genetherapy delivery and expression system according to the presentinvention. In particular, it will be shown that intra-articularexpression of proteoglycan 4 (PRG4) in mice protects against developmentof osteoarthritis (OA). The data are supported by long-term PRG4expression under the type II collagen promoter (Col2a1) in transgenicmice. Accordingly long-term expression of PRG4 does not adversely affectskeletal development but protects from developing signs of age-relatedosteoarthritis.

The protective effect is also shown in a model of post-traumaticosteoarthritis created by cruciate ligament transection (CLT). Moreover,intra-articular injection of helper-dependent adenoviral virus (HDAd)expressing PRG4 protected against the development of post-traumaticosteoarthritis when administered either before or after injury. Geneexpression profiling of mouse articular cartilage and in vitro cellstudies show that PRG4 expression inhibits the transcriptional programsthat promote cartilage catabolism and hypertrophy through theup-regulation of hypoxia inducible factor 3 alpha. Analyses of availablehuman osteoarthritis datasets are consistent with the predictions ofthis model. Hence, the data provide insight into the mechanisms forosteoarthritis development and offer a potential chondroprotectiveapproach to its treatment.

Moreover, injection of helper-dependent adenoviral vectors expressingPRG4 (HDAd-PRG4) and Il-1Ra (HDAd-Il1-Ra) in combination into joints ofwild type mice after transection of cruciate ligaments exhibitedprotective effects against osteoarthritis. Co-injection of HDAd-PRG4 andHDAd-Il1-Ra at the same dose results in a greater extent of cartilagepreservation compared to single vector injections.

As such PRG4 in single application or in combination with Il-1Ra is anovel target in chondoprotection using the helper-dependent adenoviralvectors of the invention.

Examples

Results

PRG4 Prevents Development of Age Related Osteoarthritis Changes

To investigate the long-term effect of Prg4 over-expression, theinventors generated transgenic mice expressing Prg4 under the cartilagespecific type II collagen promoter (Col2a1) (FIG. 5A). PRG4 transgenicmice expressed Prg4 ectopically in growth plate cartilage andover-express Prg4 in articular cartilage throughout development andadulthood (FIG. 5B-D). Macroscopically, the inventors detected nodifferences in growth or skeletal development in Prg4 transgenic micewhen assessed by weight (FIG. 5E). Microscopically, markers ofchondrocyte proliferation or apoptosis remained the same in Prg4 micevs. wild type mice as assessed by BrdU staining (p=n.s. in bothproliferating and resting zone of P1 chondrocytes) (FIG. 5F) and TUNELstaining (no positive signals in P1 chondrocytes) (FIG. 5G),respectively. These data suggested that ectopic over-expression of Prg4in cartilage did not significantly affect chondrocyte or skeletalhomeostasis.

The inventors sought to determine whether Prg4 over-expression inparticular chondrocytes protected mice from age-related osteoarthriticchanges. Relatively few studies have been performed to assess thedevelopment of age-related osteoarthritis in animal models (M.Silbermann, E. Livne, Age-related degenerative changes in the mousemandibular joint. Journal of Anatomy 129, 507 (October, 1979). Moreover,no gain of function model has been shown to be protective againstage-related osteoarthritis. In an aging cohort, as assessed by theOsteoarthritis Research Society International (OARSI) histologicalgrading scale (S. S. Glasson, M. G. Chambers, W. B. van den Berg, C. B.Little, The OARSI histopathology initiative—recommendations forhistological assessments of osteoarthritis in the mouse. Osteoarthritisand cartilage/OARSI, Osteoarthritis Research Society 18, S17 (Oct. 1,2010), the inventors observed that wild type FVB/N mice developedchanges consistent with moderate osteoarthritis by 10 months of age,with a mean OARSI grade of 3.5. However, PRG4 transgenic mice at thesame age exhibited a mean OARSI grade of 2 (p<0.05), suggesting lesssevere signs of osteoarthritis (FIG. 1A). The inventors next assessedthe molecular differences in particular chondrocytes between the PRG4and wild type mice. As Collagen type X (Col10a1) and matrixmetalloproteinase 13 (Mmp13) are markers of cartilage hypertrophy anddegradation, respectively, increased expression of Mmp13 and Col10a1above the tide marks are hallmarks of osteoarthritis (T. Saito et al.,Transcriptional regulation of endochondral ossification by HIF-2α duringskeletal growth and osteoarthritis development. Nature Medicine 16, 678(Jun. 23, 2010)). Encouragingly, the inventors detected increasedexpression of Col10a1 and Mmp13 in wild type mice with aging, while Prg4transgenic mice did not show a qualitative increase in either marker inthe noncalcified region of articular cartilage (FIG. 1 b ). Theseresults suggest that Prg4 overexpression had a protective effect againstosteoarthritis at the molecular and histological levels.

A disadvantage of conventional histological endpoints is the lack ofthree-dimensional quantification as well as ascertainment bias based onchoice of sections. Hence, the inventors applied an approach to quantifycartilage properties (e.g., volume, surface area, bone area covered bycartilage) based on three-dimensional reconstructions of phase contrastμCT imaging data (M. Ruan et al., Quantitative imaging of murineosteoarthritic cartilage by phase contrast micro-computed tomography.Arthritis Rheum, (2012)). Using this imaging technique, the inventorsfound that wild type mice showed a decrease in articular cartilagevolume as well as in the bone area covered by cartilage (FIG. 1C,D). Incontrast, Prg4 transgenic mice showed preservation of articularcartilage volumes and surface area (p<0.01) (FIG. 1C,D). Thus, ascompared to wild type mice after transection, Prg4 transgenic mice hadnone of the histological, molecular and imaging findings characteristicof osteoarthritis. These data suggested that PRG4 over-expression mayhave protective effects in the context of age-relatedosteoarthritic-like changes.

PRG4 Prevents Development of Post-Traumatic Osteoarthritis

To test whether PRG4 over-expression protects mice from the developmentof more aggressive, post-traumatic osteoarthritis, the inventors appliedthe knee cruciate ligament transection model recently developed in theinventors' lab, to both wild type and Prg4 transgenic mice (M. Ruan etal., Quantitative imaging of murine osteoarthritic cartilage by phasecontrast micro-computed tomography. Arthritis Rheum, (2012). Theinventors chose this approach because anterior cruciate ligament tearsare a common cause of post-traumatic arthritis in humans. As assessed bythe OARSI histological grading scale, wild type mice developed moderateand severe osteoarthritis one and two months after transection,respectively (FIG. 2A) (M. Ruan et al., Quantitative imaging of murineosteoarthritic cartilage by phase contrast micro-computed tomography.Arthritis Rheum, (2012). Prg4 transgenic mice showed a lower grade OARSIscore compared to wild type mice one and two months after transection(FIG. 2A), Interestingly, one month after transection, the OARSI gradeof cartilage from Prg4 transgenic mice was not significantly differentfrom wild type mice after sham surgery (FIG. 2A), further supportingthat PRG4 expression had a protective effect against osteoarthritis. Inaddition, the inventors detected increased expression of Col10a1 andMmp13 in the noncalcified articular cartilage of wild type transectedmice, but not in transected Prg4 transgenic mice or wild type mice aftersham surgery (FIG. 2B).

The inventors next assessed the cartilage volume and bone area coveredby cartilage after surgical transection using phase-contrast microCT (M.Ruan et al., Quantitative imaging of murine osteoarthritic cartilage byphase contrast micro-computed tomography. Arthritis Rheum, (2012)).After transection, wild type mice showed decrease in both cartilagevolume and bone area covered by cartilage (p<0.01). In contrast, Prg4transgenic mice showed articular cartilage volumes and areas similar towild type mice after sham surgery (FIG. 2C,D). Thus, as compared to wildtype mice after transection, Prg4 transgenic mice had none of thehistological, molecular or imaging findings characteristic ofosteoarthritis.

Pain and motor dysfunction are also hallmarks of osteoarthritis and aretypical causes of chronic disability (M. B. Goldling, S. R. Goldring,Osteoarthritis. Journal of Cellular Physiology 213, 626 (2007)), Theyalso serve as important clinical end points for interventional trials.Therefore, the inventors applied rodent behavioral testing, i.e.,rotarod and hotplate analyses, to evaluate for potential motor and/orsensory dysfunction in wild type vs. Prg4 transgenic mice afterosteoarthritis induction. Surgically transected wild type mice showed adecreased time on the rotarod (p<0.05) and increased time on thehotplate (p<0.05), while Prg4 transgenic mice with and withouttransection were indistinguishable from wild type mice after shamsurgery (p=n.s.) (FIG. 2E,F). This suggested that Prg4 transgenic miceafter transection had less motor or sensory impairments as compared towild type mice, supporting that PRG4 prevented functional impairment inpost-traumatic osteoarthritis.

Gene Transfer with HDAd-PRG4 Effectively Treats Osteoarthritis

To translate localized expression of PRG4 into a therapeutic approach,the inventors tested whether gene transfer into the joint could mediatelong-term expression and chondroprotection in osteoarthritis. Sincedelivery of recombinant protein is often therapeutically limited bytheir short half-life, the inventors chose to use a viral gene transferapproach. The most studied viral vectors for gene transfer related toosteoarthritis treatment are adeno-associated virus (AAV) andadenovirus. Both have been shown to transduce chondrocytes in vitro inprimary chondrocyte and cartilage organ cultures and in vivo in rabbitand rat knee joints (J. D. Kay et al., Intra-articular gene delivery andexpression of interleukin-1Ra mediated by self-complementaryadeno-associated virus. The journal of gene medicine 11, 605 (July,2009); Y. Arai et al., Gene delivery to human chondrocytes by an adenoassociated virus vector. journal of Rheumatol 27, 979 (April, 2000); J.Gouze, Adenovirus-mediated gene transfer of glutamine:fructose-6-phosphate amidotransferase antagonizes the effects ofinterleukin-1β on rat chondrocytes. Osteoarthritis and Cartilage 12, 217(April, 2004)). However, no direct comparison has been made between thetwo viruses. After injection of GFP expressing helper-dependentadenovirus and AAVs of the serotypes 2; 2.5 and 6 into mouse knee joints(10⁹ viral particles per joint in 5 ul), helper-dependent adenovirus wasnoted to exhibit higher transduction efficiency at 2 weekspost-injection (FIG. 3A).

While first generation adenovirus vectors (FGV) can mediate highlyefficient tissue transduction, the immune response to viral proteinslimits transgene expression. Previous studies performed by the inventorsand others showed that helper-dependent adenoviral vectors (HDAd) devoidof viral coding genes could overcome this problem (D. J. Palmer, D. J.P. D. P. Ng, Helper-dependent adenoviral vectors for gene therapy. Humangene therapy 16, 1 (2005)). For example, a single injection of HDAd canmediate long-term transgene expression in small and large animal modelsfor over 7 years in liver (N. Brunetti-Pierri, P. Ng, Helper-dependentadenoviral vectors for liver-directed gene therapy. Hum Mol Genet 20, R7(Jun. 13, 2011)). Thus, the inventors tested whether HDAd could mediatelong-term expression of luciferase in mouse joint compared to FGVs.Indeed, the inventors found that after a single infra-articularinjection, HDAds mediated expression of luciferase in mouse knee jointsfor over one year, while FGV-mediated luciferase expression was lost byone month (FIG. 3B). To evaluate the dose response and cellulardistribution of transduction, the inventors assessed mouse knee jointsinjected with 10⁹ vs. 10⁸ viral particles HDAd expressingbeta-galactosidase. These doses were at least 10 and 100 times lowerthan the maximum tolerated systemic dose in humans (K. Relph, K.Harrington, H. Pandha, Recent developments and current status of genetherapy using viral vectors in the United Kingdom. BMJ (Clinicalresearch ed.) 329, 839 (Oct. 9, 2004)).

At the higher dose, HDAd transduced superficial layer chondrocytes andsynoviocytes, while only synoviocytes were transduced at the lower dose(FIG. 3C). Thus, the inventors showed that HDAd was able to efficientlytransduce synoviocytes and chondrocytes with maintaining transgeneexpression for at least one year.

To compare the effects of PRG4 expression from superficial layerchondrocytes vs. synoviocytes, the inventors treated mice at both doseswith HDAd expressing PRG4 (FIGS. 6A-C) 48 hours prior to surgicalcruciate ligament transection. The inventors found that both low dose(mediating expression only in synoviocytes) and high dose (mediatingexpression in both synoviocytes and superficial zone chondrocytes)treatment with HDAd-PRG4 vector protected joints from osteoarthritisdevelopment (FIG. 3D-G). Since the clinical application of PRG4 forosteoarthritis would likely be administrated after an injury, theinventors next tested the efficacy of HDAd-PRG4 injection two weeksafter osteoarthritis induction. In this context, injection of the lowerdose of HDAd-PRG4 was sufficient to preserve cartilage volumes andprevent cartilage degradation as assessed by μCT, while higher doseinjection showed protective effects both by histological OARSI gradingand μCT assessment. Importantly, these data suggested that ectopicexpression from synoviocytes was sufficient to achieve a certain degreeof chondroprotection by acting in a non-cell-autonomous fashion (FIG.3H-K).

PRG4 Inhibits Transcriptional Programs of Chondrocyte Hypertrophy andHypoxic Inducible Factors in Cartilage

The potential mechanisms of the protective effects of PRG4 have onlybeen partially deciphered. While previous studies have shown that PRG4relieves mechanical stress in joints by changing synovial fluid dynamicsand providing boundary lubrication (G. D. Jay, J. R, Torres, M. L.Warman, M. C. Laderer, K. S. Breuer, The role of lubricin in themechanical behavior of synovial fluid. Proc Natl Acad Sci USA 104, 6194(Apr. 10, 2007)), the inventors investigated whether PRG4 could directlyaffect cartilage metabolism and homeostasis. To assess the moleculareffects of PRG4 on chondrocytes, the inventors performed transcriptionalprofiling on superficial layer chondrocytes obtained by laser capture innewborn wild type vs. Prg4 transgenic mice (FIG. 4A and FIG. 7A). Genesthat were either up-regulated or repressed by greater than 1.5 fold(Dataset 1) were analyzed by Ingenuity Pathway Analysis to identifytranscriptional programs that would be affected by Prg4 expression(Dataset 2). Interestingly, transcription factors that mediatechondrocyte hypertrophy and terminal differentiation (e.g. Mef2c, Runx2and Atf4) showed decreased activity with PRG4 over-expression (K. S. Leeet al., Runx2 Is a Common Target of Transforming Growth Factor beta 1and Bone Morphogenetic Protein 2, and Cooperation between Runx2 andSmad5 Induces Osteoblast-Specific Gene Expression in the PluripotentMesenchymal Precursor Cell Line C2C12. Molecular and cellular biology20, 8783 (Dec. 1, 2000); X. Yang et al., ATF4 is a substrate of RSK2 andan essential regulator of osteoblast biology; implication forCoffin-Lowry Syndrome. Cell 117, 387 (May 30, 2004); M. A. Arnold etal., MEF2C Transcription Factor Controls Chondrocyte Hypertrophy andBone Development. Developmental Cell 12, 377 (April, 2007)). Inaddition, the inventors also noted down-regulation of Smad7, aninhibitor of the TGFbeta signaling pathway (A. Nakao et al.,Identification of Smad7, a TGFbeta-inducible antagonist of TGF-betasignalling. Nature 389, 631 (Oct. 9, 1997)). TGFbeta signalingnegatively regulates terminal differentiation of chondrocytes, andhence. PRG4 again would suppress hypertrophy by its actions on Smad7 (X.Yang et al., TGF-beta/Smad3 signals repress chondrocyte hypertrophicdifferentiation and are required for maintaining articular cartilage.The Journal of cell biology 153, 35 (May 2, 2001)). Finally, Hypoxiainducible factor 1 alpha unit (Hif1alpha), an essential transcriptionfactor induced in response to hypoxia, was also predicted to have loweractivity (FIG. 4B), while Hif3alpha a post-translational negativeregulator of Hif1alpha and Hif2alpha was up-regulated (Y. Makino et al.,Inhibitory PAS domain protein is a negative regulator ofhypoxia-inducible gene expression. Nature 414, 550 (Nov. 29, 2001)).(FIG. 4A and FIG. 7B).

The inventors hypothesized that PRG4 could up-regulate Hif3alpha underhypoxic conditions to inhibit cartilage turnover. This effect would bemediated by down-regulating the Hif1alpha and Hif2alpha transcriptionalactivities. To test our hypothesis, the inventors measured Hif3alphaexpression and downstream Hif target genes relevant to osteoarthritisprogression under hypoxic conditions in C3H10T1/2 (mesenchymal stromal)cells. After injection of HDAd-PRG4, Hif3alpha was transcriptionallyup-regulated while Vegf, Col101a1 and Mmp13, all markers of hypertrophy,were all down-regulated compared to empty vector (FIG. 4C) (T. Saito etal., Transcriptional regulation of endochondral ossification by HIF-2αduring skeletal growth and osteoarthritis development. Nature Medicine16, 678 (Jun. 23, 2010)). Next, the inventors assessed whether knockdown of Hif3alpha suppressed the effects caused by over-expression ofPRG4. Since the expression of Hif3alpha is low in C3H10T1/2 cells, theinventors tested its expression in alternative chondrogenic lines.Interestingly, compared to under normoxic conditions, all cell linestested showed increased PRG4 expression under hypoxic conditions (FIG.4D and FIG. 7C). Consistent with this, Genomatix analysis identified ahypoxia response element in the promoter region of PRG4. The inventorsfound that Hif3alpha was highly expressed in TC71 Ewing sarcoma cellsand further increased with up-regulation of PRG4 under hypoxicconditions (FIG. 4D). As predicted by our previous transcriptomicanalysis, knockdown of Hif3alpha in TC71 cells led to down regulation ofHif1alpha and Hif2alpha target genes in the face of PRG4 up regulation(FIG. 4E). Our findings suggested that the secreted protein PRG4 couldmodify the balance of anabolic and catabolic programs in the context ofosteoarthritis pathogenesis.

To investigate whether the signalling pathway discovered in mouse isconserved in humans, the inventors performed in silico analysis on geneexpression profiling performed in human osteoarthritis patient samplesavailable from the GEO database (S, Koelling et al., MigratoryChondrogenic Progenitor Cells from Repair Tissue during the Later Stagesof Human Osteoarthritis, Stem Cell 4, 324 (May 3, 2009); T, Dehne, C.Karlsson, J. Ringe, M. Sittinger, A. Lindahl, Chondrogenicdifferentiation potential of osteoarthritic chondrocytes and theirpossible use in matrix-associated autologous chondrocytetransplantation. Arthritis research &amp; therapy 11, R133 (2009)). Theinventors discovered PRG4 and the proposed downstream effector,HIF3alpha, are upregulated in chondrocyte progenitor cells in OApatients by 2.6 fold (p<0.05) and 1.5 fold (p<0.01) respectively. In anindependent array set comparing 3 dimensional cultured chondrocytes fromosteoarthritis and healthy donors, the inventors observed a similartrend: PRG4 was upregulated by 1.4 fold (p<0.05) and HIF3alphaupregulated by 1.3 fold (p<0.05). In the context of osteoarthritisdevelopment, PRG4 and H1F3alpha may both be upregulated as a repairresponse. In contrast to the sustained over expression of PRG4 in ourtherapeutic models, this normal response in humans may be insufficientto prevent disease progression.

These data together showed that under the hypoxic conditions ofcartilage, PRG4 over-expression may prevent osteoarthritis progressionnot only by exerting biomechanical effects on the synovial fluid andcartilage interface, but also by regulating the transcriptional networksthat specify chondrocyte hypertrophy and catabolism. Cartilage turnovermediated by Hif1alpha and Hif2alpha was inhibited by up-regulation ofHif3alpha. As cartilage degradation and hypertrophy are two hallmarks ofosteoarthritis progression, it is not surprising that PRG4 haschondroprotective effects both in age-related and post-injuryosteoarthritis (FIG. 4F).

Osteoarthritis Gene Therapy can be Enhanced by Combined Gene Transfer ofPRG4 and Il-1Ra

The inventors sought to evaluate whether the beneficial effect ofover-expressing PRG4 in osteoarthritis joints can be further improved bycombining it with gene therapy mediated expression of Il-1Ra. Il-1Rablocks the effects of Il-1beta, which is one of the key drivers ofinflammation and cartilage catabolism in osteoarthritis. Based on thedifferent pathways that PRG4 and Il-1Ra exert their effects on, acombination of both might result in optimized inhibition of bothcartilage breakdown and inflammation.

Mice had osteoarthritis induced and were injected with gene therapyvectors two weeks later. HDAd-PRG4. HDAd-Il-1Ra and the combination ofboth resulted in significantly lower osteoarthritis histology scorescompared to the control vector HDAd-GFP and the no treatment group (FIG.8A). Cartilage surface area was significantly higher in the HDAd-PRG4,HDAd-Il-1Ra and combination group compared with HDAd-GFP (FIG. 8B).Furthermore, cartilage volume was significantly higher in the HDAd-PRG4,HDAd-Il-1Ra and combination group compared with HDAd-GFP (FIG. 8C). Thecombination of HDAd-PRG and HDAd-Il-1Ra resulted in significantly highercartilage volume compared with single vector treatment. The combinationtherapy resulted also in lower average osteoarthritis score and higheraverage cartilage surface area; however, the difference in theseparameters did not reach statistical significance although a trend tosignificance was present.

Discussion

The invention shows by using both transgenic mice expressingProteoglycan 4 (PRG4), and intra-articular, helper-dependent adenoviralvirus (HDAd) gene transfer that PRG4 is protective against thedevelopment of both post-traumatic and age-related osteoarthritis,without significant adverse effects on cartilage development. Genetherapy treatment with HDAd-PRG4 was effective when injected before andafter onset of osteoarthritis suggesting that the treatment is bothpreventive and therapeutic. The beneficial effect can be furtherimproved by combining PRG4 with anti-inflammatory Il-1Ra gene therapy.The protective effects are demonstrated at molecular, histological andfunctional levels. The inventors further show that PRG4 over-expressioninhibits transcriptional programs that promote cartilage catabolism andhypertrophy in part through the up-regulation of Hif3alpha. Theconcordant changes of PRG4 and HIF3alpha expression is also observed ingene expression profiling in human osteoarthritic patient samples.

Most genetics models reported to date show protection fromosteoarthritis using histological endpoints at one month after surgicaldestabilization of the medical meniscus (DMM) to induce a mild, singlecondylar post-traumatic osteoarthritis, In addition, studies onosteoarthritis have been largely focused on loss of function mutationsof genes in bone development such as Adamts5, Mmp13, Hif2alpha andSyndecan4 (F. Echtermeyer et al., Syndecan-4 regulates ADAMTS-5activation and cartilage breakdown in osteoarthritis. Nature Medicine, 1(Mar. 30, 2102); T. Saito et al., Transcriptional regulation ofendochondral ossification by HIF-2α during skeletal growth andosteoarthritis development. Nature Medicine 16, 678 (Jun. 23, 2010); S,S. Glasson et al., Deletion of active ADAMTS5 prevents cartilagedegradation in a murine model of osteoarthritis. Nature 434, 644 (Apr.31, 2005); C. B. Little et al., Matrix metalloproteinase 13-deficientmice are resistant to osteoarthritic cartilage erosion but notchondrocyte hypertrophy or osteophyte development. Arthritis &Rheumatism 60, 3723 (December, 2009)). In contrast to these studies, theinventors report the gain of function genetic model with a secretedprotein PRG4 that protects against osteoarthritis development at least 2months after transection of cruciate ligaments, This model mimics acommon injury in humans and leads to osteoarthritis in both condylarstructures of the knee. The establishment of a gain of function modelusing an endogenously produced secreted protein may make for easierclinical translation as compared to previous approaches targetinginhibition of specific matrix enzymes and/or intracellular transcriptionfactors. Moreover, the demonstration of a beneficial effect onage-related cartilage changes supports the further study of thisapproach beyond injury model.

The established mechanisms that protect animals from osteoarthritisdevelopment mostly depend on inhibition of cartilage catabolic enzymes.ADAMTS5 was the first target to be discovered via in vivo geneticexperiments (S. S. Glasson et al., Deletion of active ADAMTS5 preventscartilage degradation in a murine model of osteoarthritis. Nature 434,644 (Apr. 31, 2005)). Loss of Syndecan 4, similarly, works throughADAMTS5 inhibition (F. Echtermeyer et al., Syndecan-4 regulates ADAMTS-5activation and cartilage breakdown in osteoarthritis. Nature Medicine, 1(Mar. 30, 2102)). Recently, the discovery of the protective effects ofHif2alpha loss of function in OA extends this approach as Hif2alphatranscriptionally regulates the expression of catabolic enzymesincluding several MMPs and ADAMTSs (T. Saito et al., Transcriptionalregulation of endochondral ossification by HIF-2α during skeletal growthand osteoarthritis development. Nature Medicine 16, 678 (Jun. 23,2010)). However, targeting anabolic pathways, including cell growth,differentiation and matrix synthesis, is equally important inosteoarthritis since chondrocyte proliferation, metaplasia and abnormalmatrix synthesis have been long observed in osteoarthritis progression(K. P. Pritzker et al., Osteoarthritis cartilage histopathology: gradingand staging. Osteoarthritis Cartilage 14, 13 (January, 2006)). Aninteraction between cartilage anabolic and catabolic pathways isrequired to maintain homeostasis and their imbalance leads toosteoarthritis progression. A therapy that can affect both programswould potentially be most effective.

Low-grade inflammation is commonly observed in osteoarthritic joints(Felson D T. 2006. Clinical practice. Osteoarthritis of the knee. N EnglJ Med 354:841-848). Besides maintaining and amplifying inflammation, thekey inflammatory mediators in osteoarthritis such as Il-1beta alsotrigger the expression of cartilage degrading enzymes such ascollagenases and aggrecanases (Daheshia, M., and YAO, J. Q. (2008). TheInterleukin 1p Pathway in the Pathogenesis of Osteoarthritis. JRheumatol 35, 2306). Therefore, it seems important to inhibit bothcartilage catabolism and joint inflammation in order to achieveefficient osteoarthritis treatment. Along these lines, the inventorsshow here that a gene therapy treatment combining helper-dependentadenoviral vectors expressing PRG4 and the anti-inflammatory Il-1Raseems to further improve osteoarthritis treatment over gene therapy withPRG4.alone.

Materials and Methods

Generation of transgenic mice. FVB/N mice were purchased from JacksonLaboratories (Bar Harbor, Me.). This strain is the common backgroundstrain for transgenic mouse lines. All studies were performed withapproval from the Baylor College of Medicine Institutional Animal Careand Use Committee (IACUC). All mice were housed under pathogen-freeconditions in less than five per cage. Mice had free access to feed andwater. Transgenic mice were generated by pronuclear microinjection.Founders were outcrossed for at least 3 generations to eliminatemultiple insertions. Different lines were tested at the beginning torule out position effect. Genotyping primers were designed to detect theVVPRE element in the transgene cassette: F: TCTCTTTATGAGGAGTTGTGGCCC(SEQ ID NO: 40), R: CGACAACACCACGGAATTGTCAGT (SEQ ID NO: 41). To avoidthe effects of potential post-menopausal bone loss, all the mice used inOA evaluation were males.

Cruciate ligament transection (CLT) surgery. CLT surgery and sham wereperformed as previously described in 8-week old male FVB/N mice and PRG4transgenic mice (M. Ruan et al., Quantitative imaging of murineosteoarthritic cartilage by phase contrast micro-computed tomography.Arthritis Rheum, (2012). Investigators were blinded to the genotype ofthe mice when surgery was performed.

Histology and immunohistochemistry. Mice were euthanized and sampleswere fixed with 4% paraformaldehyde (Sigma-Aldrich) overnight in 4° C.on a shaker. Samples from mice older than 4 days were decalcified in 14%EDTA for 5 days in 4° C. on a shaker. Samples from mice younger than 3days were not decalcified. Paraffin embedding was performed aspreviously described. Samples were sectioned at 6 μm. Samples werestained with safranin O and fast green using standard protocols. Sampleswere scored by two independent pathologists masked to the procedure andgenotypes. Immunohistochemistry were performed using primary antibody:anti-PRG4 (Abcam, ab 28484), anti-MMP13 (Millipore, MAB 13424),anti-ColX (generous gift from Dr. Greg Lunstrum, Shriners Hospital forChildren, Portland, Oreg.), and secondary antibody: one-dropper-bottleHRP polymer conjugates (Invitrogen). BrdU staining was performed usinganti-BrdU Alexa Fluor 594 (A21304, Invitrogen). Histomark trueblue (KPL)was used as developing reagent. TUNEL staining was performed usingApopTag Plus Peroxidase In situ Apoptosis Detection (Millipore KitS7101) following manufacturer's protocol. All staining in the sameexperiment were done at the same time. Observer who quantified of BrdUand TUNEL staining was blinded to the genotype of the mice.

Beta-galactosidase staining. Staining was performed on samples embeddedin optimal cutting temperature compound after fixation anddecalcification. Samples were sectioned at 6 μm and stained with X-gal(X428IC Gold biotechnology) overnight and nuclear fast red (N3020 Sigma)as counter stain.

Rotarod analysis. Mice were placed onto an accelerating rotarod (UGOBasile, Varese, Italy). The duration to first failure to stay atop therod was marked as first ride-around time. To rule out differences inlearning skills between the two groups of mice, each group was assessedover three trials per day for 2 consecutive days (trials 1 to 6) beforesurgery. Mice were then randomly assigned into different groups. Another6 trials were performed using the same conditions at the different timepoints after the surgery. Mice were given a 30 minutes inter-trial restinterval. Each trial had a maximum time of 5 minutes. Observer wasblinded to the genotype and the procedure of the mice.

Hotplate analysis. Mice were placed on the hotplate at 55° C. (ColumbusInstruments, Columbus, Ohio). The latency period for hind limb response(e.g. shaking, jumping, or licking) was recorded as response time beforeat different time points after surgery. Observer was blinded to thegenotype and the procedure of the mice.

Phase contrast μCT scanning. Samples were prepared as previouslydescribed and scanned by Xradia μXCT at source voltage=40 kV, sourcepower=8 W, detector distance from sample=75 mm, source distance fromsample=100 mm, image number taken=500, and exposure time for eachimage=30 (M. Ruan et al., Quantitative imaging of murine osteoarthriticcartilage by phase contrast micro-computed tomography. Arthritis Rheum,(2012)). The resolution of the scanning is 4 μm. After scanning, arandom number was assigned to each sample to ensure blinded assessmentduring image processing.

Reconstruction and analysis of μCT data. Reconstruction of the data wasperformed using Xradia software and was transformed into dicom files.Reconstruction involves correction for beam hardening (constant=0.3),and correcting for center shift effects caused by difference between thecenter of sample rotation and the center of the detector. Samples wereanalyzed using TriBON software (RATOC, Tokyo, Japan). Observers wereblinded to the procedure and sample number (M. Ruan et al., Quantitativeimaging of murine osteoarthritic cartilage by phase contrastmicro-computed tomography. Arthritis Rheum, (2012)).

Intra-articular Injection. Mice were anesthetized using 3% isoflurane.Joint area was shaved. HDAds were diluted in sterile PBS in 5 μl andinjected by 25 μl CASTIGHT syringes (1702 Hamilton Company) and 33 gaugeneedles (7803-05 Hamilton Company).

Luciferase assay. Mice were injected with 2 mg D-luciferin (L9504 SIGMA)diluted in 100 μl PBS per mouse (25 grams) intraperitoneally. Mice wereanesthetized using 3% isoflurane. Images were taken by Xenogen IVISoptical in vivo imaging system. Quantification was performed by livingImaging 4.2 using default settings. Image was collected for 10 minutesafter the injection and normalized to control mice without luciferaseinjection.

Laser capture microdissection and RNA purification. Hind limbs of P1littermates were collected and snap-frozen in liquid nitrogen. Then,samples were embedded in optimal cutting temperature compound. Frozensections of 10 μm were generated on polyethylene napthalate(PEN)-membrane slides. Superficial layer chondrocytes were capturedusing HS Capsure LCM caps by Applied Biosciences Acturus Systems. RNAwas then purified by Picopure RNA isolation kit.

Mouse Microarray and analysis. Microarrays were performed using MouseWG-6 v2.0 Expression BeadChip (Illumina). Data was processed using thelumi package within the R statistical package. Variance-stabilizingtrans-formation (VST) was performed, followed by quantile normalizationof the resulting expression values. Differential expression wascalculated using the limma package within R. Heat map was generatedusing normalized fold change. The resulting lists were then annotatedand reviewed for candidates.

Human gene expression analysis. GEO archives GSE10575 titled “Migratorychondrogenic progenitor cells from repair tissue during the later stagesof human osteoarthritis” (PMID: 19341622) and GSE16464, titled“Chondrogenic differentiation potential of osteoarthritis chondrocytesand their use in autologous chondrocyte transplantation” (PMID:19723327) were both downloaded and analyzed using the web-based GEO2R,using the default settings, available through the GEO site. In archiveGSE10575, three arrays of chondrogenic progenitor cells fromosteoarthritis males were compared to two control arrays of the samecell type. Female samples were excluded because control samples aremales (shown by the level of Xist expression). In archive GSE 16464,3D-cultured chondrocytes from normal donors and 3D-cultured chondrocytesfrom OA donors were compared. Both archives used the Affymetrix HumanGenome U133 Plus 2.0 Array platform.

Cell culture, transfection and infection. C3H10T1/2 cells weremaintained in DMEM with 10% FBS; TC71 cells were maintained in RPMI 1640with 10% FBS; ATDC5 cells were maintained in DMEM/F-12 1:1 mixturesupplemented with 10% FBS. Cells were plated the day beforetransfection/infection so that it reached 70% confluency the next day.Lipofectamine 2000 was used as transfection reagent following protocolsprovided by manufacturer. Dharmacon on-target siRNA was used in theknockdown assay. HDAds were generated as previously described (M. Suzukiet al., Large-scale production of high-quality helper-dependentadenoviral vectors using adherent cells in all factories. Human genetherapy 21,120 (February, 2010)). HDAd-PRG4 carries the murine PRG4 genecontrolled by the constitutive EF1 promoter. HDAd-Il-1Ra carries themurine Il-1Ra gene controlled by an inflammation-inducible NF-κBpromoter. To infect cells. HDAds were diluted at 5000 vp/cell and addedin serum free media with minimal volume covering cells after aspiration.Two hours later, media containing virus was aspirated and culturingmedia was added back. For hypoxia experiments, cells were transferred tohypoxia chamber with 1% oxygen.

RNA purification and quantitative PCR. Cells were lysed with Trizolreagents (Invitrogen) and RNA was purified following manufacturer'sprotocol. To eliminate DNA contamination, samples were treated withRNase-free recombinant DNaseI (Roche). Reverse-transcript PCR wasconducted by superscript III first strand (18080-051Invitrogen)following manufacturer's protocol. Taqman Universal PCR mastermix(Applied biosciences) and PerfeCTa SYBR Green SuperMix (QuantaBioSciences) were used in quantitative PCR. Primers used in quantitativePCR are listed as follows: mouse

F: (SEQ ID NO: 16) ACTTCAGCTAAAGAGACACGGAGT, R: (SEQ ID NO: 17)GTTCAGGTGGTTCCTTGGTTGTAGTAA; Sox9: F: (SEQ ID NO: 18)AAGCCACACGTCAAGCGACC, R: (SEQ ID NO: 19) GTGCTGCTGATGCCGTAACT; Col2a1:F: (SEQ ID NO: 20) GCTCATCCAGGGCTCCAATGATGTAG, R: (SEQ ID NO: 21)CGGGAGGTCTTCTGTGATCGGTA; Gapdh: F: (SEQ ID NO: 22)GCAAGAGAGGCCCTATCCCAA; R: (SEQ ID NO: 23) CTCCCTAGGCCCCTCCTGTTATT; Vegf:F: (SEQ ID NO: 24) TGGACTTGTGTTGGGAGGAGGATG, R: (SEQ ID NO: 25)GCCTCTTCTTCCACCACCGTGTC; Mmp13: F: (SEQ ID NO: 26)GCAATCTTTCTTTGGCTTAGAGGT, R: (SEQ ID NO: 27) GGTGTTTTGGGATGCTTAGGGT;Col10a1: F: (SEQ ID NO: 28) AAAGCTTACCCAGCAGTAGG, R: (SEQ ID NO: 29)ACGTACTCAGAGGAGTAGAG; GAPDH: F: (SEQ ID NO: 30)ATACCAGGAAATGAGCTTGACAAA, R: (SEQ ID NO: 31) TGAAGGTCGGAGTCAACGGA; VEGF:F: (SEQ ID NO: 32) GATCGGTGACAGTCACTAGCTTATCT, R: (SEQ ID NO: 33)TACACACAAATACAAGTTGCCA; MMP13: F: (SEQ ID NO: 34)TGCCCTTCTTCACACAGACACTAACGAAA, R: (SEQ ID NO: 35)GGCCACATCTACTATTCTTACCACTGCTC; COL10A1: F: (SEQ ID NO: 36)GCCCACTACCCAAGACCAAGAC; R: (SEQ ID NO: 37) GACCCCTCTCACCTGGACGAC; HIF3A:F: (SEQ ID NO: 38) GGCTGTTCCGCCTACGAGTA; R: (SEQ ID NO: 39)AGCAAGGTGGATGCTCTTG; PRG4: Hs00981633_m1 (applied biosciences);mouse Hif3a: Mm00469375_m1 (applied bicsciences).

Statistics. Statistical significance comparing two groups withparametric data was assessed by Student's t test, Statistical analysiscomparing multiple groups with parametric data was performed by one-wayANOVA followed by Tukey's post-hoc. Statistical analysis comparingdifferent genotype with different procedure was performed by two-wayANOVA followed by Tukey's post-hoc, Normality was tested by Shapiro-WilkNormality test. Histological grades were compared by Wilcox rank test.All analyses were performed by SPSS software or Sigma Plot. A P value of<0.05 was considered statistically significant.

Dataset 1: List of genes showing more than 1.5 fold change in themicroarray analysis. WTP1_1 WTP1_2 WTP1_3 PG4P1_1 PG4P1_2 PG4P1_34930546H06Rik_ILMN_2717117 1.256363 1.360731 1.425929 −1.48447 −1.59768−1.52108 Sesn1_ILMN_2654074 −1.25604 −1.26072 −1.28391 1.234927 1.1704781.226371 Ypel5_ILMN_1251071 1.224517 1.294585 1.311664 −1.44517 −1.32632−1.38253 Bglap-rs1_ILMN_1233122 1.301868 2.023561 1.628096 −3.0338−3.05449 −2.56761 AK038070_ILMN_2466021 1.257255 1.181603 1.204158−1.30391 −1.20282 −1.31808 AK011460_ILMN_2452717 −1.52912 −1.85077−1.48354 1.474372 1.352955 1.304319 Bglap1_ILMN_2610166 1.4041372.037433 1.6235 −4.09687 −3.29889 −2.57926 Med18_ILMN_1214050 −1.48231−1.26813 −1.3871 1.212726 1.267995 1.335163 Bglap1_ILMN_3101908 1.4591172.042785 1.496434 −3.82431 −3.03438 −2.43533 Ppp1r3c_ILMN_2667091−1.65083 −1.56199 −1.48727 1.4376 1.167941 1.476123Bglap-rs1_ILMN_1220829 1.261849 2.094587 1.691356 −3.64532 −3.48459−2.55816 Zfp46_ILMN_1215740 −1.35114 −1.26299 −1.37143 1.277394 1.1367891.32476 Hist1h1c_ILMN_2855315 1.196666 1.423895 1.348674 −1.49333−1.34167 −1.62396 Zfand2a_ILMN_1230489 1.273059 1.140929 1.34758−1.36636 −1.26743 −1.39361 Ddx21_ILMN_2546724 1.305544 1.217222 1.179125−1.34811 −1.20085 −1.382 Hist1h1c_ILMN_2774537 1.170861 1.3844531.428754 −1.41392 −1.42079 −1.65332 Ddx54_ILMN_2689678 1.130087 1.2866181.299302 −1.39565 −1.2461 −1.30723 Rps15a_ILMN_2717621 1.315098 1.1390161.449143 −1.36561 −1.52841 −1.40807 Bglap-rs1_ILMN_2944508 1.3039822.185122 1.520279 −3.70241 −3.45533 −2.31957 Slc7a5_ILMN_27119481.201827 1.326767 1.430059 −1.67471 −1.48398 −1.29808Csnk1d_ILMN_2739965 1.11045 1.255629 1.292397 −1.37356 −1.22945 −1.24982Dyrk1b_ILMN_3053158 −1.46521 −1.31006 −1.19201 1.182241 1.2948581.238163 Hist2h3b_ILMN_2934120 1.254913 1.119073 1.304197 −1.38431−1.299 −1.20538 Nmd3_ILMN_1228859 1.262366 1.122484 1.224622 −1.20301−1.36721 −1.20793 1190005F20Rik_ILMN_2697918 −1.21616 −1.40512 −1.447451.36511 1.269442 1.140632 Lrrc59_ILMN_1252817 1.334493 1.222121 1.168151−1.47501 −1.29518 −1.21186 Dusp8_ILMN_1228031 1.288724 1.069707 1.275318−1.27037 −1.24915 −1.28446 AK053260_ILMN_1228804 −1.53513 −1.52115−1.78747 1.594074 1.424237 1.113431 Eif1a_ILMN_2698107 1.399002 1.3480681.07882 −1.44405 −1.34553 −1.35426 AK021349_ILMN_1258961 1.3828041.270095 1.173873 −1.34656 −1.59361 −1.2452 Csnk1d_ILMN_1231035 1.1049831.305878 1.354114 −1.47277 −1.23751 −1.33697 Cirbp_ILMN_2761594 1.086571.233789 1.298241 −1.23605 −1.35285 −1.20021 Plekhf2_ILMN_27986941.124677 1.171659 1.32521 −1.2892 −1.34384 −1.16463 Nfatc1_ILMN_12165221.286293 1.080945 1.308693 −1.21468 −1.39646 −1.27436 Clk1_ILMN_2428301−1.36643 −1.38464 −1.9415 1.450046 1.301662 1.279182 Trove2_ILMN_12527251.195356 1.139589 1.272322 −1.22692 −1.41043 −1.15117EG639396_ILMN_2877059 −1.19236 −1.20876 −1.39289 1.317211 1.119031.179859 Heatr1_ILMN_1214036 1.317403 1.264432 1.223294 −1.64477−1.32574 −1.20107 BC027809_ILMN_1252263 1.357622 1.055984 1.444709−1.34453 −1.47672 −1.38744 Ccdc130_ILMN_2756733 −1.17101 −1.26787−1.41093 1.342647 1.169009 1.136904 Tmcc1_ILMN_1249710 −1.26174 −1.39677−1.70812 1.477478 1.22952 1.199067 Arrdc4_ILMN_2648967 1.045261 1.5742521.593893 −1.83439 −1.7299 −1.50742 AK044963_ILMN_1247942 1.3216981.257721 1.270333 −1.18779 −1.38985 −1.69824 Arf2_ILMN_1214810 1.5460191.353128 1.087012 −1.4972 −1.66445 −1.34205 2610101N10Rik_ILMN_1252490−1.14039 −1.40709 −1.51568 1.290925 1.162057 1.299667Lrrc47_ILMN_2628551 1.117726 1.123233 1.383986 −1.31904 −1.23011 −1.2438Srm_ILMN_2809611 1.485302 1.196046 1.150938 −1.54798 −1.39998 −1.23852Gprc5a_ILMN_2854943 1.320606 1.042922 1.353049 −1.29647 −1.25915−1.39294 Ddx11_ILMN_2700550 −1.20131 −1.24547 −1.33501 1.353463 1.2046011.057541 Deb1_ILMN_2652971 1.548657 1.086404 1.190474 −1.36664 −1.38753−1.38497 Mif_ILMN_2867835 1.125532 1.187658 1.327057 −1.3999 −1.32759−1.12086 Hoxd3_ILMN_1219807 −1.19061 −1.30954 −1.55302 1.334377 1.3163371.101846 Cdc7_ILMN_1238374 −1.31242 −1.26666 −1.86596 1.253473 1.2810071.378172 Zfp187_ILMN_3067831 −1.18727 −1.40885 −1.54414 1.4576551.221198 1.121467 3300001P08Rik_ILMN_2727004 −1.16879 −1.39116 −1.322351.224787 1.064016 1.380551 Hoxc6_ILMN_1217328 −1.33051 −1.47422 −1.400751.339994 1.496705 1.019485 Rbm5_ILMN_2942492 −1.29365 −1.62199 −1.684641.66198 1.236204 1.11868 AK012053_ILMN_2469320 −1.10834 −1.3099 −1.403481.32378 1.110387 1.187654 Tmem128_ILMN_1248235 1.125965 1.1385221.441919 −1.45467 −1.29997 −1.19488 Rbm5_ILMN_2942499 −1.45228 −1.59992−1.84238 1.607189 1.534819 1.001613 E130016E03Rik_ILMN_3161959 −1.2783−1.50572 −1.71101 1.568022 1.337492 1.063614 Tnfrsf12a_ILMN_24242991.680884 1.277593 1.075987 −1.78329 −1.46531 −1.38442 Prdm2_ILMN_12504541.173017 1.20632 1.256779 −1.14469 −1.19644 −1.52795 Srm_ILMN_12258801.332372 1.120064 1.166016 −1.4506 −1.22663 −1.14034 Cpt2_ILMN_2775123−1.20532 −1.33083 −1.38724 1.104396 1.131549 1.462135 Twf1_ILMN_12442191.25757 1.126966 1.235596 −1.49415 −1.20089 −1.13911AK076052_ILMN_2429108 1.076837 1.140505 1.40668 −1.19618 −1.25719−1.34308 Farsa_ILMN_1257639 1.213878 1.216622 1.352934 −1.12429 −1.38604−1.65116 Zfp84_ILMN_2506757 −1.16964 −1.18995 −1.49011 1.301893 1.1049541.226727 Rnase4_ILMN_1235657 −1.46403 −1.24206 −1.12261 1.3239191.163284 1.133851 Pim3_ILMN_2717667 1.123427 1.144501 1.541166 −1.42121−1.21509 −1.50536 Rab5b_ILMN_1237467 −1.54934 −1.50901 −1.79875 1.8241541.27382 1.037959 Tk1_ILMN_2605890 −1.14797 −1.19726 −1.52316 1.2001721.303368 1.13359 Nrp1_ILMN_1247094 −1.34674 −1.14618 −1.60363 1.2446151.403199 1.113605 6030458C11Rik_ILMN_1234196 −1.5397 −1.45778 −2.222281.702852 1.461039 1.050668 Pold1_ILMN_2655577 −1.24471 −1.24541 −1.47811.425738 1.253099 1.038272 Prss35_ILMN_2609897 1.480951 −1.038321.608742 −1.44299 −1.64698 −1.54549 Tppp3_ILMN_2655929 −1.08048 −1.39071−1.3892 1.218015 1.318692 1.098879 Clec2d_ILMN_2603647 −1.73457 −1.2971−2.52661 1.280941 1.696474 1.279334 Hif3a_ILMN_2649671 −1.78312 −1.6431−1.32223 1.73224 1.313779 1.028263 Pnrc2_ILMN_2861331 −1.12969 −1.18773−1.58246 1.161941 1.19852 1.280471 Myd116_ILMN_2722938 1.38863 1.1490011.09759 −1.13863 −1.23126 −1.4829 Eln_ILMN_2697304 1.09603 1.3104371.235098 −1.20273 −1.1346 −1.54888 Fbxo31_ILMN_2452855 −1.16259 −1.1719−1.62377 1.23252 1.296053 1.142115 Zmym2_ILMN_2781493 −1.1322 −1.205−1.65697 1.259404 1.17403 1.249941 Rad54l_ILMN_2741985 −1.14435 −1.14233−1.57512 1.210268 1.2324 1.17319 Nedd9_ILMN_2654186 1.300471 1.3692141.006802 −1.27054 −1.16693 −1.47168 Gadd45g_ILMN_2744890 1.1598731.093824 1.401321 −1.50495 −1.10425 −1.29047 Syncrip_ILMN_12584201.212463 1.238146 1.1686 −1.42916 −1.42482 −1.02121 Ier3_ILMN_12167641.881239 1.019378 1.168521 −1.48139 −1.66306 −1.52784 Galt_ILMN_2677567−1.21625 −1.36275 −1.40341 1.540977 1.172451 1.018011Map3k1_ILMN_2614380 −1.22523 −1.25855 −1.72004 1.286516 1.4798161.041544 Rrp12_ILMN_2728118 1.330091 1.265982 1.182798 −1.73253 −1.45231−1.0467 Dbp_ILMN_2616226 −1.3989 −1.2558 −2.17137 1.654516 1.1664721.207322 Sfrs7_ILMN_2552490 −1.09319 −1.37266 −1.50612 1.421972 1.2255561.045249 Tmem100_ILMN_1224014 −1.53254 −1.20266 −1.67189 1.4995281.458731 −1.04208 Rpap1_ILMN_1238065 −1.22594 −1.19869 −1.41188 1.4803941.120412 1.040978 AK017419_ILMN_2416218 1.258403 −1.04994 1.597254−1.40368 −1.39738 −1.30912 Trpc2_ILMN_1220948 −1.1478 −1.20737 −1.487021.435121 1.116418 1.076493 Il11ra1_ILMN_1229957 −1.17181 −1.34116−1.36512 1.51528 1.033071 1.120113 AK007736_ILMN_1258587 −1.30198−1.1931 −1.5084 1.02934 1.151676 1.549816 Atpbd4_ILMN_1230688 1.2121651.044319 1.518026 −1.1629 −1.29912 −1.67838 Tuba1c_ILMN_2476139 1.1851761.22582 1.255489 −1.65104 −1.03071 −1.31991 Abhd14b_ILMN_3007862−1.17428 −1.31793 −1.82363 1.555108 1.207257 1.078925Ifi27l2a_ILMN_2762944 1.023728 1.619691 1.164394 −1.58706 −1.19828−1.37445 Mll5_ILMN_1217776 −1.08848 −1.35305 −1.70073 1.363244 1.3450821.045908 AK029312_ILMN_1246692 −1.1321 −1.22047 −1.47253 1.301738−1.00965 1.326047 Cars2_ILMN_1218543 −1.15407 −1.26682 −1.75735 1.4716861.249823 1.053571 Raver2_ILMN_1213278 −1.20699 −1.23934 −1.940091.342784 1.066234 1.440154 Mid1_ILMN_3159435 1.127098 1.038972 1.460931−1.13274 −1.25072 −1.44792 AK078053_ILMN_1222351 −1.10161 −1.27506−1.55656 1.45516 1.108898 1.101466 AK028672_ILMN_1246030 1.2237921.161685 1.206628 −1.02194 −1.2642 −1.56654 AK043421_ILMN_2575994−1.26016 −1.2699 −2.19263 1.481949 1.406903 1.07406AK085118_ILMN_2505392 −1.11638 −1.20699 −1.48486 1.360363 1.2325081.009408 Lpp_ILMN_2463260 1.144334 1.172656 1.289494 1.004724 −1.44657−1.43369 NR_001461_ILMN_2445958 −1.33534 −1.58378 −1.69795 1.8617531.221821 −1.05574 Zfp52_ILMN_2838139 −1.25077 −1.21913 −1.67899 1.5448611.244823 −1.00507 AK004187_ILMN_2513451 −1.30128 −1.18771 −1.301721.256082 1.421114 −1.05914 Gp1bb_ILMN_2653205 1.310991 −1.01749 1.499583−1.20277 −1.26174 −1.71629 BC037034_ILMN_1257019 −1.07956 −1.19392−1.58424 1.197539 1.32752 1.079839 AK086317_ILMN_2580737 1.9742061.266016 1.059287 −1.17028 −2.25587 −2.48319 NR002848_ILMN_2966602−1.34056 −1.63363 −1.01451 1.281704 1.267341 1.107163 Asns_ILMN_26435131.12627 1.082326 1.473384 −1.63603 −1.22191 −1.12562AK005089_ILMN_2451115 1.054101 1.319016 1.421071 −1.13881 −1.24881−1.89777 6430706D22Rik_ILMN_3011719 −1.33993 −1.32228 −1.58013 1.6228331.345146 −1.11534 5330401P04Rik_ILMN_2520011 −1.1508 −1.17966 −1.631071.427003 1.221601 1.021642 Flcn_ILMN_1213483 −1.17513 −1.1274 −1.555131.410583 1.179933 1.028483 Per2_ILMN_2987862 −1.21694 −1.06498 −1.654281.267989 1.067504 1.299293 Ppm1m_ILMN_1224437 −1.16175 −1.17068 −1.911211.437044 1.159707 1.165048 AK084113_ILMN_2451389 −1.16976 −1.13767−1.70942 1.283512 1.371085 1.026543 AK007605_ILMN_1239776 1.1198951.118125 1.47604 −1.14506 −1.18395 −1.76057 Depdc6_ILMN_3163001 −1.13115−1.20253 −1.78263 1.08135 1.165515 1.47653 Mterf_ILMN_2624809 −1.0454−1.20426 −1.5987 1.344071 1.147785 1.095675 AK014695_ILMN_2748880−1.23553 −1.24466 −2.42903 1.564719 1.31189 1.098905 Vat1l_ILMN_1226356−1.17602 −1.20391 −1.46333 1.144397 −1.02032 1.511196Ankzf1_ILMN_2703321 −1.20097 −1.19701 −1.82856 1.447617 1.36439 −1.02771Tha1_ILMN_2594768 −1.09096 −1.25311 −1.78214 1.402023 1.303303 1.018911Plekhf1_ILMN_2993334 −1.08624 −1.16026 −1.78844 1.297336 1.2427471.118284 Gprasp1_ILMN_3142384 −1.15055 −1.14972 −1.66918 1.0144351.224737 1.422807 Pdgfra_ILMN_1235932 −1.28478 −1.3484 −1.50982 1.627191.311287 −1.13736 Mcm10_ILMN_2970532 −1.0688 −1.14651 −1.70048 1.1611081.296402 1.146574 Fbp2_ILMN_2634905 −1.67939 −1.76924 −2.20592 −1.362061.508826 2.142995 1110007M04Rik_ILMN_2734060 1.125411 1.216839 1.439906−1.77901 −1.60341 1.032062 Suv420h2_ILMN_1260420 −1.06155 −1.32955−2.05337 1.396444 1.349127 1.073276 Adat2_ILMN_2705097 1.042499 1.0800971.530343 −1.07116 −1.36781 −1.46542 1200016B10Rik_ILMN_1236716 −1.08228−1.17359 −1.66229 1.410154 1.112488 1.099721 AK021262_ILMN_2546861−2.55735 −2.96551 −2.04609 2.589175 1.589228 −1.65394NR_002848_ILMN_2438819 −1.04157 −1.19208 −1.62472 1.102605 1.1303351.352612 Iqcb1_ILMN_2635348 −1.25858 −1.51247 −1.07587 1.479027 1.1129911.022781 Sirpa_ILMN_2722996 −1.55085 1.020695 −1.42422 1.095271 1.3978551.139231 Kif1b_ILMN_2587761 −1.10748 −1.26856 −1.71852 −1.03298 1.2759941.482789 Gtpbp2_ILMN_2600113 −1.03362 −1.24214 −1.75238 1.3140341.280253 1.062528 Akap8l_ILMN_1242769 −1.70634 −1.79935 −1.468551.863393 1.552434 −1.31333 AK029270_ILMN_1246021 −1.36249 −1.13168−2.29819 1.675332 1.15085 1.121103 Timm8a1_ILMN_2896552 1.1839761.065901 1.33591 −1.5964 −1.26281 −1.0041 Cpt2_ILMN_2775122 −1.31568−1.57523 −1.10064 −1.0034 1.133167 1.566763 Mif_ILMN_1260512 1.0049751.323347 1.316609 −1.60996 −1.34456 −1.0099 Cenpl_ILMN_2676726 −1.07561−1.16457 −1.74209 1.300334 1.282306 1.05495 Ccdc86_ILMN_2730003 1.0796311.174875 1.493109 −1.81679 −1.39094 −1.01727 Cited2_ILMN_2477221−1.25941 −1.07229 −1.54732 −1.01603 1.188502 1.45439 Tle6_ILMN_2900617−1.25696 −1.29766 −2.43442 1.763195 1.23855 1.021292 Mrm1_ILMN_2649654−1.12291 −1.18885 −1.88148 1.496945 1.102948 1.136915Tsc22d3_ILMN_3150811 −1.46991 −1.23229 −1.52848 1.715752 1.257691−1.13571 Wnk1_ILMN_1234955 −1.1436 −1.18321 −1.62288 1.476736 1.220566−1.03422 Gstt3_ILMN_2665715 −1.15761 −1.16266 −1.67614 1.531124 1.0532151.095113 Ppm1k_ILMN_2923615 −1.08558 −1.15334 −1.64931 1.404428 1.1349971.066046 Clk4_ILMN_2851710 −1.15318 −1.33127 −1.79737 1.595963 1.307868−1.08522 Il11ra2_ILMN_2619594 −1.04918 −1.28561 −1.57792 1.4750521.023734 1.136498 Cars2_ILMN_2670601 −1.09189 −1.14083 −1.62876 1.3890331.161988 1.042614 Pbx1_ILMN_2559669 −1.2673 −1.25299 −2.278 1.7033661.314599 −1.04615 Acot11_ILMN_1227579 −1.16373 −1.10948 −1.738311.422989 1.008933 1.232184 Neat1_ILMN_2493030 −1.1433 −1.74898 −4.340581.651763 1.528512 1.142921 AK020467_ILMN_2506727 −1.09908 −1.81523−1.30434 1.576618 1.226821 −1.03184 Calb2_ILMN_2827729 −1.13166 −1.24465−1.58857 1.188467 1.548766 −1.05688 Rassf4_ILMN_2956092 −1.19773−1.02283 −1.71608 1.100194 1.347195 1.157291 Tia1_ILMN_1215055 −1.07031−1.11087 −1.74002 1.144017 1.141023 1.305753 Csnk2a1_ILMN_1218670−1.22304 −1.30599 −2.00138 1.579869 1.464052 −1.14536 INV_ILMN_1257729−1.19494 −1.45406 −1.54665 1.780692 1.124428 −1.08257Fam109a_ILMN_2668178 −1.1457 −1.10631 −2.07282 1.126329 1.3716241.242875 Clspn_ILMN_2858359 −1.09104 −1.12459 −2.01152 1.193798 1.2112731.292024 AK051059_ILMN_2419748 −1.0608 −1.16991 −1.93243 1.3797571.164043 1.141268 Unc5c_ILMN_2461668 −1.01081 1.174419 1.441474 −1.35249−1.00552 −1.51306 AK078921_ILMN_2462678 −1.11083 −1.23212 −1.740511.561371 1.188618 −1.03775 Sgk_ILMN_1213954 1.274151 1.55087 −1.15069−1.29135 −1.183 −1.4572 Disp1_ILMN_2772288 −1.08894 −1.41679 −1.494691.632533 1.126658 −1.05526 Gadd45g_ILMN_2903945 1.451953 1.3184141.04743 −2.13919 1.014627 −1.42835 LOC100040259_ILMN_1244853 −1.183131.187774 1.974228 −1.83552 −1.42003 −1.34451 _ILMN_1245646 −1.029561.218253 1.413033 −1.59167 −1.26363 −1.02272 Tmem129_ILMN_2429215−1.09374 −1.11964 −2.07634 1.200101 1.167903 1.342939 Tusc4_ILMN_2454195−1.08831 −1.10728 −1.85511 1.369108 1.179966 1.089908Map3k12_ILMN_2725370 −1.12888 −1.35818 −2.18302 1.726495 1.263127−1.07506 Ndrg2_ILMN_2771991 −1.07046 −1.1445 −2.06977 1.291192 1.0954541.322285 Rrp15_ILMN_2629856 1.004134 −1.02887 1.835562 −1.43385 −1.48925−1.22032 Mum1_ILMN_1215647 −1.06142 −1.13778 −1.74536 1.418322 1.0837031.103988 AK052106_ILMN_1255302 −1.05526 −1.20509 −1.60772 1.399531.257665 −1.06004 Tlcd1_ILMN_2781458 −1.02375 −1.3448 −2.10692 1.4759821.34724 −1.0186 Trps1_ILMN_1226073 −1.06444 −1.5275 −1.78979 1.7574461.159398 −1.07492 Ly6a_ILMN_1255416 −3.17206 −1.0932 −1.29711 1.4617671.331041 1.206246 Accs_ILMN_2776485 −1.22008 −1.009 −1.65779 1.417321.13865 1.030122 Appl2_ILMN_1219978 −1.24559 −1.27531 −2.11094 1.8651221.161517 −1.09567 Cox4i2_ILMN_2612178 1.089127 1.158668 1.3269571.008061 −1.18331 −1.74794 Clk1_ILMN_1254814 −1.07911 −1.22534 −2.015591.568467 1.193925 −1.00131 Clspn_ILMN_2623056 −1.05233 −1.08879 −1.827611.147235 1.150539 1.28634 Ncrna00166_ILMN_1222196 −1.03988 −1.16428−1.98635 1.39944 1.094827 1.181747 C4b_ILMN_3049559 −1.10606 −1.15706−2.05407 1.351722 1.414119 −1.0215 Nfatc4_ILMN_2647331 −1.37338 −1.25155−1.20467 1.5856 1.183728 −1.14491 AK036974_ILMN_1222598 −1.0959 −1.17652−1.59093 1.5362 1.079376 −1.00664 Slc5a3_ILMN_1233078 −1.17885 −1.23164−1.89385 1.499145 1.458686 −1.17105 Abcd4_ILMN_1245547 −1.16716 −1.03031−1.70307 1.428297 1.110164 1.047001 Spnb2_ILMN_1214394 −1.0152 −1.12231−1.88513 1.152147 1.231002 1.210335 Prelp_ILMN_2739760 −1.11189 −1.53571−1.7425 1.801632 1.224774 −1.17762 5830411K21Rik_ILMN_1217032 −1.39544−1.00477 −1.90468 1.069172 1.653072 1.04086 Bmp4_ILMN_1215252 −1.23614−1.17177 −1.52917 1.595397 1.230579 −1.16592 Hoxd8_ILMN_2693052 −1.07822−1.07856 −1.81581 1.155206 1.051639 1.387814 1810013L24Rik_ILMN_2616630−1.18506 1.006997 −2.02702 1.342216 1.132491 1.181125430432N15Rik_ILMN_2622089 −1.48775 −1.75547 1.115626 1.119898 1.1194611.403214 Stxbp3a_ILMN_1245393 −1.41728 −1.33413 −1.13258 1.5773821.23735 −1.18035 Ehd1_ILMN_2628757 −1.17445 1.186862 1.650685 −1.45395−1.2074 −1.25788 Raf1_ILMN_1237730 −1.07621 −1.19935 −1.61396 1.52391.171328 −1.08435 Tbc1d2b_ILMN_2819859 −1.07046 −1.08129 −2.172661.290826 1.221243 1.168665 Ppox_ILMN_2826816 −1.06553 −1.21576 −1.960911.443881 1.371369 −1.09439 Eraf_ILMN_2619200 −1.14473 1.597198 1.416693−2.1079 1.035463 −1.65928 Hbb-b1_ILMN_1244316 1.271854 1.441461 1.216162−2.80858 1.193409 −1.91916 Chst5_ILMN_2665754 1.01614 −1.33756 −1.724231.103901 −1.00597 1.558293

Dataset 2: List of transcription factors predicted by Ingenuity PathwayAnalysis to be activated or inhibited. Positive z score suggestsactivation and negative z score. Transcription Regulation p-valueRegulator z-score of overlap Target molecules in dataset Molecular TypePPARA 2.196 1.89E−01 ASNS, CHKA, Clec2d (includes ligand-dependentothers), CPT2, FABP3, HIST1H1C, IGFBP5, nuclear receptor KIF2C, LGALS4,RETSAT, SRM, TOP2A STAT5B 2.038 1.42E−01 MYL2, TNNC1, TNNT1, TPM3,TROVE2 transcription regulator PPARD 2.036 1.29E−01 ACTG2, CPT2, FABP3,FN1, LGALS4, ligand-dependent TNFRSF12A nuclear receptor ESR1 2.0213.42E−01 BMP4, Clec2d (includes others), DDX21, ligand-dependent HOXC6,IER3, IGFBP5, SGK1, TGFB3 nuclear receptor FOXO3 −2.188 2.97E−01 IER3,PPP1R15A, SGK1, SLC1A4 transcription regulator MEF2C −2.359 2.06E−02Bglap (includes others), IBSP, MYL2, TNNC1 transcription regulator FOS−2.58 3.37E−01 CASZ1, CDON, FN1, HBA1/HBA2, HBB, IBSP, transcriptionregulator IGFBP5, KIF1B, LGALS4, NFATC1, S100A8, S100A9, SIRPA GATA4−2.64 1.61E−02 ACTC1, ACTG2, MYL4, MYLPF, TNNC1 transcription regulatorMYOCD −2.976 1.88E−04 ACTG2, GJA5, LPP, MYL2, MYL4, MYLPF transcriptionregulator SRF −3.454 1.05E−03 ACTC1, ACTG2, GADD45G, HIF3A, LBH,transcription regulator MYL2, MYL3, MYL4, Myl9, MYLPF, RAF1, TNNC1,TNNT1, ZMYM2 GLI1 1.845 1.78E−01 LMNA, NDRG2, PDGFRA, S100A9, TMEM100,transcription regulator WIF1 E2F1 1.844 3.83E−01 BMP4, DDX11/DOX12P,MCM10 transcription regulator (includes EG: 307126), NRP1 (includes EG:18186), POLD1, PRDM2, RAD54L, TK1, TOP2A SATB1 0.865 3.41E−02 ABTB1,HBB, HSPA8, SGK1, TSC22D3, transcription regulator YPEL5 CEBPD 0.7953.70E−02 ASNS, IGFBP5, MBP, MIA, PDGFRA transcription regulator TFAP2C0.427 3.11E−03 HIST1H1C, MBP, NRP1 (includes transcription regulator EG:18186), TK1, ZMYND11 SMARCB1 0.271 3.27E−02 C4B (includes others), CDC7(includes transcription regulator EG: 12545), KIF23, MCM10 (includes EG:307126), PLXNB2, PPP1R3C, RAB5B, RAD54B RUNX2 −0.09 7.90E−03 ACTG2,Bglap (includes others), C4B transcription regulator (includes others),COL24A1, FN1, IBSP SMAD7 −0.265 4.16E−02 ACTG2, CITED2, FN1, MYLPF,TGFB3, TPM3 transcription regulator PGR (includes −0.275 5.84E−03 CPT2,DDX21, IER3, IGFBP5, NEDD9, ligand-dependent EG: 18667) SRSF7, TGFB3,TK1, TSC22D3 nuclear receptor TP53 (includes −0.709 1.68E−03 ASNS, BMP1,CCDC80, CDC7 (includes transcription regulator EG: 22059) EG: 12545),CSNK1D, DBP, FABP3, FN1, GADD45G, GSTM1, HDC, HJURP, HK2, HSPA8, IER3,IGF3P5, IKBIP, IQCB1, KIF23, LPP, Ly6a (includes others), MYL4, Myl9,NDRG2, NRP1 (includes EG: 18186), PDGFRA, PEG3, PLXNB2, POLD1, PPP1R15A,PQLC3, RAD54B, RAF1, RNASE4, SESN1, SGK1, TOP2A, TSC22D3 CEBPB (includes−1.133 2.42E−02 ACTG2, ARPP19, ASNS, CIRBP, Gnas transcription regulatorEG: 1051) (mouse), HBB, HDC, IER3, MBP, MIA, NRP1 (includes EG: 18186),PDGFRA, PPP1R15A, SGK1 MITF −1.373 3.65E−02 CHKA, CMA1, EDNRB, GPNMB,MBP, MYL4, transcription regulator Tpsab1 KDM5B −1.459 1.71E−02 EHD1,IARS2, KIF2C, NEDD9, PPOX, transcription regulator PSIP1, TOP2A GATA1−1.697 1.36E−02 AHSP, ALAS2, GP1BB, HBA1/HBA2, HBB, transcriptionregulator MBP HIF1A −1.714 3.60E−03 ACTG2, ASNS, CHKA, CITED2, FN1,HIF3A, transcription regulator HIST1H1C, HK2, IGFBP5, MIF, PFKL, SC29A1,TGFB3, TMEM128 SMARCA4 −1.791 9.94E−03 ACTG2, ASNS, Bglap (includesothers), transcription regulator BMP4, CLK1, FN1, HBB, IGFBP5, LMNA,MYH3, MYL4, MYLPF, NRP1 (includes EG: 18186) MYOD1 −1.839 6.25E−03ACTC1, ACTG2, GADD45G, IGFBP5, MYH3, transcription regulator MYL4,MYLPF, SPT8N1, TNNC1, TNNT1 ATF4 −1.848 7.63E−03 ASNS, Bglap (includesothers), IBSP, transcription regulator IGFBP5, PPP1R15A, SLC7A5,TNFRSF12A

SEQUENCE LISTING OVERVIEW

SEQ ID NO 1 HDAd-huPRG4

SEQ ID NO 2 HDAd-muPRG4

SEQ ID NO 3 huPRG4

SEQ ID NO 4 muPRG4

SEQ ID NO 5 huPRG4 protein

SEQ ID NO 6 muPRG4 protein

SEQ ID NO 7 HDAd-hulL1 Ra

SEQ ID NO 8 HDAd-mulI1 Ra

SEQ ID NO 9 HDAd-eqlI1 Ra

SEQ ID NO 10 huIl1Ra

SEQ ID NO 11 muIl1 Ra

SEQ ID NO 12 eqIl1Ra

SEQ ID NO 13 huIl1 Ra protein

SEQ ID NO 14 muIl1 Ra protein

SEQ ID NO 15 eqIl1Ra protein

hu=human; mu=murine; eq=equine

The invention claimed is:
 1. A pharmaceutical composition, comprising atherapeutically effective amount of at least one helper-dependentadenoviral vector containing a nucleic acid sequence encodingproteoglycan 4 (PRG4), or a biologically active fragment thereof, whichhas chondoprotective activity, a left and a right adenoviral invertedterminal repeats (L ITR and R ITR), adenoviral packaging signalsequences and non-viral, non-coding stuffer nucleic acid sequences,wherein i. the at least one helper-dependent adenoviral vectoradditionally comprises a nucleic acid sequence encoding interleukin-1receptor antagonist (II-1Ra), or ii the composition comprises a secondhelper-dependent adenoviral vector comprising a nucleic acid sequenceencoding Il-1Ra; wherein PRG4 expression is controlled by an elongationfactor 1 alpha (EF 1 alpha) promoter and expression of Il-1Ra iscontrolled by an NF-κB promoter, and wherein the at least onehelper-dependent adenoviral vector containing a nucleic acid sequenceencoding PRG4, comprises a nucleic acid sequence which has at least 90%sequence homology with the nucleic acid sequence set forth in SEQ ID NO:1, or SEQ ID NO:
 2. 2. The pharmaceutical composition according to claim1, wherein the nucleic acid sequence encoding PRG4 comprises a nucleicacid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or abiologically active fragment thereof, or a homolog thereof from anyother species, or a nucleic acid sequence which has at least 90%sequence homology with a nucleic acid sequence set forth in SEQ ID NO 3,or SEQ ID NO 4, or a biologically active fragment thereof, wherein thePRG4 encoded by the biologically active fragment of SEQ ID NO 3, or SEQID NO 4, or a nucleic acid sequence which has at least 90% sequencehomology with a nucleic acid sequence set forth in SEQ ID NO 3, or SEQID NO 4, or a biologically active fragment thereof, has chondoprotectiveactivity.
 3. The pharmaceutical composition according to claim 1,wherein the amino acid sequence of PRG4 comprises an amino acid sequenceset forth in SEQ ID NO 5, or SEQ ID NO 6, or a biologically activefragment thereof, or a homolog thereof from any other species, or anamino acid sequence which has at least 90% sequence homology with anamino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or abiologically active fragment thereof, wherein the amino acid sequence ofPRG4, which comprises a biologically active fragment of the amino acidSEQ ID NO 5, or SEQ ID NO 6, or has at least 90% sequence homology withan amino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or abiologically active fragment thereof, has chondoprotective activity. 4.The pharmaceutical composition according to claim 1, wherein the atleast one helper-dependent adenoviral vector comprising the nucleic acidsequence encoding Il-1Ra, comprises a nucleic acid sequence which has atleast 90% sequence homology with a nucleic acid sequence set forth inSEQ ID NO 7, or SEQ ID NO 8, or SEQ ID NO 9, or a biologically activefragment thereof, wherein the at least one helper-dependent adenoviralvector comprising the nucleic acid sequence encoding II-1Ra, whichcomprises a nucleic acid sequence which has at least 90% sequencehomology with a nucleic acid sequence set forth in SEQ ID NO 7, or SEQID NO 8, or SEQ ID NO 9, or a biologically active fragment thereof, hasII-1Ra activity of inhibiting inflammatory and cartilage destructivemediators.
 5. The pharmaceutical composition according to claim 1,wherein the nucleic acid sequence encoding Il-1Ra comprises a nucleicacid sequence set forth in SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO12, or a biologically active fragment thereof, or wherein the nucleicacid sequence encoding for Il-1Ra comprises a nucleic acid sequencewhich has at least 90% sequence homology with a nucleic acid sequenceset forth in SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, or abiologically active fragment thereof, wherein the Il-1Ra encoded by thebiologically active fragment of SEQ ID NO 10, or SEQ ID NO 11, or SEQ IDNO 12, or the nucleic acid sequence which has at least 90% sequencehomology with a nucleic acid sequence set forth in SEQ ID NO 10, or SEQID NO 11, or SEQ ID NO 12, or the biologically active fragment thereof,mediates II1-Ra activity of inhibiting inflammatory and cartilagedestructive mediators.
 6. The pharmaceutical composition according toclaim 1, wherein the amino acid sequence of Il-1Ra comprises the aminoacid sequence of human IL-1Ra (SEQ ID NO 13), murine IL-1Ra (SEQ ID NO:14), equine IL-1Ra (SEQ ID NO: 15), or a biologically active fragmentthereof, or a homolog thereof from any other species, which has Il-1Raactivity of inhibiting inflammatory and cartilage destructive mediators.